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Are there plasma membrane receptors for thyroid hormones? | Receptors for thyroid hormones are present on plasma membrane of cells; in particular thyroid hormones bind integrin that is a heterodimeric component of plasma membrane | The thyrotropin receptor from bovine thyroid plasma membranes has been
solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin
binding to the solubilized receptor has been developed. Both the solubilized
thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes
have effectively identical nonlinear Scatchard plots and negatively sloped Hill
plots, i.e. both preparations have receptors which appear to exhibit a similar
negatively cooperative relationship. Although the pH optimum of thyrotropin
binding to the solubilized receptor is the same as that of the thyroid plasma
membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor
is slightly different in its outline. Thyrotropin binding to the solubilized
receptor is less sensitive to salt inhibition than is binding to the thyroid
plasma membrane receptor; however, optimal binding remains at 0 degrees. The
relative affinities of thyrotropin and two glycoprotein hormones which can be
considered structural analogs, luteinizing hormone and human chorionic
gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but
100:25:40 toward the solubilized receptors. The solubilized receptor preparation
is heterogeneous in size in that it has binding components with molecular
weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion
converts all three higher molecular weight components to the 15,000 to 30,000
molecular weight species, and the 15,000 to 30,000 molecular weight receptor
component has all of the binding properties of the solubilized receptor
preparation before tryptic digestion including an identical nonlinear Scatchard
plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to
30,000 molecular weight receptor released by tryptic digestion of bovine thyroid
plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture.
The tryptic fragment of the solubilized receptor or preparations has been
purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose
columns. The binding activity is lost when the solubilized thyrotropin receptor
preparation is exposed to beads of neuraminidase-Sepharose or conconavalin
A-Sepharose. Comparative and competitive analyses of thyroxine (T4) and triiodothyronine (T3)
binding to highly purified rat liver, brain and lung cell plasma membranes were
carried out. The dependence of hormone binding on the time, temperature and
concentration was studied. The effects of trypsin and partial delipidation on
the binding parameters of thyroid hormones were investigated. Two thyroid
hormone-binding sites were detected in cell plasma membranes of all tissues
under study. The maximal binding of T4 to rat liver membranes and the maximal
binding of T3 to rat brain membranes was observed in all experiments, the
affinity for T3 being higher than that for T4. An important role of both protein
and lipid components of plasma membranes in the membrane reception of thyroid
hormones is proposed. In this presentation, I present evidence indicating a direct action of thyroid
hormone at the level of the plasma membrane. Characteristically, the plasma
membrane-mediated effects of thyroid hormones are prompt in onset, independent
of new protein synthesis, and are associated with changes in the transmembrane
transport of ions and substrates. The presence of specific binding sites for
thyroid hormone in plasma membrane of various tissues and species, although
inconclusive in itself, provides additional support for the direct action of
thyroid hormone on the plasma membrane. A model for the mechanism of action of
thyroid hormone at the plasma membrane level to increase sugar uptake by rat
thymocytes is delineated, and the physiological role of the plasma
membrane-mediated action of thyroid hormone is discussed. The in vivo translocation of thyroxine-binding blood serum prealbumin (TBPA) was
studied. It was found that the TBPA-hormone complex penetrates-through the
plasma membrane into the cytoplasm of target cells. Electron microscopic
autoradiography revealed that blood serum TBPA is localized in ribosomes of
target cells as well as in mitochondria, lipid droplets and Golgi complex.
Negligible amounts of the translocated TBPA is localized in lysosomes of the
cells insensitive to thyroid hormones (spleen macrophages). Study of T4- and
T3-binding proteins from rat liver cytoplasm demonstrated that one of them has
the antigenic determits common with those of TBPA. It was shown
autoimmunoradiographically that the structure of TBPA is not altered during its
translocation. The binding characteristics of thyroxine (T4), triiodothyronine (T3), and
reverse T3 (rT3) to rat liver plasma membranes (RLPM) were examined to explore
the interactions of thyroid hormones with cell surface receptors. Scatchard
analysis suggested that all three ligands bound to two classes of binding sites.
The high affinity rT3 binding sites appeared to be distinct from the high
affinity T4 sites, on the basis of differing optimum physicochemical conditions
for binding, and analog displacement studies. The higher affinity constant for
T4 was 1.7 +/- 0.2 X 10(9) M-1 (mean +/- SEM) and binding capacity was 3.1 +/-
0.3 pmol mg-1 protein whereas for rT3 binding the Ka was 2.5 +/- 0.4 X 10(8) M-1
and capacity was 6.2 +/- 0.9 pmol mg-1. (125I) T3 bound with lower affinity and
T3 tracer was readily displaced by T4. Moreover, comparatively higher
concentrations of T3 were needed to displace either radiolabeled T4 or rT3,
suggesting that T3 was binding to both the T4 and rT3 sites with lower affinity.
Marker enzyme studies on RLPM, of varying purity prepared by different methods,
showed a positive correlation between the activity of the plasma membrane enzyme
magnesium-stimulated ATPase and high affinity rT3 and T4 binding. Column
chromatography of the radioligands, after dissociation from membrane binding
sites, confirmed that the integrity of the hormones was not altered during
association or dissociation. Our results raise the possibility that the high
affinity T4 and rT3 binding sites on RLPM may be hormone receptors mediating
biological actions at the membrane level. Nongenomic actions of thyroid hormone are by definition independent of nuclear
receptors for the hormone and have been described at the plasma membrane,
various cell organelles, the cytoskeleton, and in cytoplasm. The actions include
alterations in solute transport (Ca2+, Na+, glucose), changes in activities of
several kinases, including protein kinase C, cAMP-dependent protein kinase and
pyruvate kinase M2 (PKM2), effects on efficiency of specific mRNA translation
and mRNA t1/2, modulation of mitochondrial respiration, and regulation of actin
polymerization (promotion of formation of F-actin). Iodothyronines also can
regulate nongenomically the state of contractile elements in vascular smooth
muscle cells (VSMC). The physiologic significance at the cellular level of
certain of these actions has been demonstrated, for example, in the cases of
myocardiocyte Na+ current, red cell Ca2+ content, and the control by
hormone-induced alterations in actin solubility of cell surface activity of
iodothyronine 5'-monodeiodinase activity and the intracellular distribution of
protein disulfide isomerase activity. The physiologic significance of these
actions at the organ or system level is less clear, but extranuclear effects of
thyroid hormone on myocardial Na+ channel, sarcoplasmic reticulum Ca(2+)-ATPase
activity, and contractile state of VSMC may each contribute to acute effects of
thyroid hormone on cardiac output that have recently been described clinically.
The molecular mechanisms for nongenomic actions are incompletely understood;
relevant binding sites and signal transduction pathways have been described for
hormone actions on plasma membrane Ca(2+)-ATPase activity, and PKM2 monomer is
known to bind T3 and, as a result, prevent activation of the kinase via tetramer
formation. Nongenomic actions of thyroid hormone may have different
structure-activity relationships of iodothyronines from those effects that
depend upon nuclear receptors; they may have different time courses and may
invoke complex signal transduction pathways before the action is detected. The presence of thyrotropin receptors (TSHR) has been reported in some
extrathyroidal tissues but its physio-pathological role still remains unclear.
TSH (seems to) affects the human erythrocytes Na(+)/K(+)-ATPase in vitro,
however receptors on erythrocytes have not yet been described. In this work the
effect of recombit human TSH (rhTSH) on sites number and activity of
erythrocyte Na(+)/K(+)-ATPase was investigated in a group of thyroidectomized
patient enrolled for rhTSH test. As detected by (3)H-ouabain binding, rhTSH
administration induced a significant increase in the number of sites (p=0.005)
and in the Kd (p=0.006) of Na(+)/K(+)-ATPase. rhTSH did not induce significant
difference in Na(+)/K(+)-ATPase activity measured by (86)Rb uptake. (125)I-TSH
binding studies and Western blotting data showed the existence of TSHR in the
erythrocytes of healthy donors. In conclusion The TSH action on
Na(+)/K(+)-ATPase of human erythrocytes can be explained by the presence of
TSHR. The nongenomic actions of thyroid hormone require a plasma membrane receptor or
nuclear receptors located in cytoplasm. The plasma membrane receptor is located
on integrin alphaVbeta3 at the Arg-Gly-Asp recognition site important to the
binding by the integrin of extracellular matrix proteins. l-Thyroxine (T(4)) is
bound with greater affinity at this site than 3,5,3'-triiodo-l-thyronine (T(3)).
Mitogen-activated protein kinase (MAPK; ERK1/2) transduces the hormone signal
into complex cellular/nuclear events including angiogenesis and tumor cell
proliferation. Acting at the integrin receptor and without cell entry, thyroid
hormone can foster ERK1/2-dependent serine phosphorylation of nuclear thyroid
hormone receptor-beta1 (TRbeta1) and de-repress the latter. The integrin
receptor also mediates actions of the hormone on intracellular protein
trafficking and on plasma membrane ion pumps, including the sodium/protein
antiporter. Tetraiodothyroacetic (tetrac) is a T(4) analog that inhibits binding
of iodothyronines to the integrin receptor and is a probe for the participation
of this receptor in cellular actions of the hormone. Tetrac blocks thyroid
hormone effects on angiogenesis and cancer cell proliferation. Acting on a
truncated form of nuclear TRalpha1 (TRDeltaalpha1) located in cytoplasm, T(4)
and 3,3',5'-triiodothyronine (reverse T(3)), but not T(3), cause conversion of
soluble actin to fibrous (F) actin that is important to cell motility, e.g., in
cells such as glia and neurons. Normal development of the central nervous system
requires such motility. TRbeta1 in cytoplasm mediates action of T(3) on
expression of certain genes via phosphatidylinositol 3-kinase (PI 3-K) and the
protein kinase B/Akt pathway. PI 3-K and, possibly, cytoplasmic TRbeta1 are
involved in stimulation by T(3) of insertion of Na,K-ATPase in the plasma
membrane and of increase in activity of this pump. Because ambient thyroid
hormone levels are constant in the euthyroid intact organism, these nongenomic
hormone actions are likely to be contributors to basal rate-setting of
transcription of certain genes and of complex cellular events such as
angiogenesis and cancer cell proliferation. Besides the well-characterized genomic action of thyroid hormone (TH), mediated
by thyroid hormone receptors (TRs), accumulating data support the so-called
non-genomic action of TH, which is often related to activation of signalling
pathways. In this study, we sought to determine whether TH activates
intracellular signalling pathways in the adult cardiac myocytes and whether such
activation modulates cell growth and the expression of target proteins important
in cardiac function. We demonstrate that TH promotes a rapid increase in the
phosphorylation of several kinases, ERK1/2, PKCdelta, p38-MAPK and Akt. This
activation is inhibited by triiodothyroacetic acid (triac), which is a TH
analogue known to displace the hormone from membrane bound receptors, indicating
that this TH effect is mediated through a cell membrane-initiated mechanism.
Furthermore, using specific inhibitors of the TH-activated kinases, we show that
the long-term effects of TH on the expression of sarcoplasmic reticulum
Ca(2+)-ATPase (SERCA), alpha- and beta-myosin heavy chain (MHC) and cell growth
are reverted, implying that what is initiated as a non-genomic action of the
hormone interfaces with genomic effects. These data provide further insights
into the underlying mechanisms of TH action in the heart with potentially
important implications in the management of cardiac pathology. Thyroid hormones play a wide range of important physiological activities in
almost all organism. As changes in these hormones levels-observed in
hypothyroidism and hyperthyroidism-promote serious derangements of the
cardiovascular system, it is important to know their mechanisms of action.
Although the classic genomic actions which are dependent on interaction with
nuclear receptors to modulate cardiac myocytes genes expression, there is
growing evidence about T(3) and T(4)-triggered nongenomic pathways, resulted
from their binding to plasma membrane, cytoplasm, or mitocondrial receptors that
leads to a rapidly regulation of cardiac functions. Interestingly both actions
converge to amplify thyroid hormone effects on cardiovascular system. T(3) and
T(4) nongenomic actions modify inotropic and chronotropic effects, cardiac
action potential duration, cardiac growth, and myocyte shape by protein
translation through protein kinases-dependent signaling cascades, which include
PKA, PKC, PI3K, and MAPK, and changes on ion channels and pumps activity. In
respect to the decreased systemic vascular resistance seen in hyperthyroidism,
T(3) appears to activate NOS or ATP-sensitive K(+) channels. In addition, a
novel biologically active T(4)-derived metabolite has been described,
3-iodothyronamine, T(1)AM, which also acts through membrane receptors to mediate
nongenomic cardiac effects. This metabolite influences the physiological
manifestations of thyroid hormone actions by inducing opposite effects from
those stimulated by T(3) and T(4), such as negative inotropic and chronotropic
effects. Therefore, beyond genomic and nongenomic effects of thyroid hormones,
it is crucial for there to be an equilibrium between T(3) or T(4) and T(1)AM
levels for maintaining cardiac homeostasis. It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and
3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response.
Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen
species, and cytokine synthesis and release, are altered in hypo- and
hyper-thyroid conditions, even though for many immune cells no clear correlation
has been found between altered levels of T(3) or T(4) and effects on the immune
responses. Integrins are extracellular matrix proteins that are important
modulators of many cellular responses, and the integrin αvβ3 has been identified
as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma
membrane binding site appears to be responsible for many nongenomic effects of
thyroid hormones, independent of the classic nuclear receptors. Through the
integrin αvβ3 receptor the hormone can activate both the ERK1/2 and
phosphatidylinositol 3-kinase pathways, with downstream effects including
intracellular protein trafficking, angiogenesis and tumor cell proliferation. It
has recently become clear that an important downstream target of the thyroid
hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New
results demonstrate the capability of T(3) or T(4) to induce in the short time
range important responses related to the immune function, such as reactive
oxygen species production and cell migration in THP-1 monocytes. Thus thyroid
hormones seem to be able to modulate the immune system by a combination of rapid
nongenomic responses interacting with the classical nuclear response. Our understanding of thyroid hormone action has been substantially altered by
recent clinical observations of thyroid signaling defects in syndromes of
hormone resistance and in a broad range of conditions, including profound mental
retardation, obesity, metabolic disorders, and a number of cancers. The
mechanism of thyroid hormone action has been informed by these clinical
observations as well as by animal models and has influenced the way we view the
role of local ligand availability; tissue and cell-specific thyroid hormone
transporters, corepressors, and coactivators; thyroid hormone receptor (TR)
isoform-specific action; and cross-talk in metabolic regulation and neural
development. In some cases, our new understanding has already been translated
into therapeutic strategies, especially for treating hyperlipidemia and obesity,
and other drugs are in development to treat cardiac disease and cancer and to
improve cognitive function. BACKGROUND: Six known steps are required for the circulating thyroid hormone
(TH) to exert its action on target tissues. For three of these steps, human
mutations and distinct phenotypes have been identified.
SCOPE OF REVIEW: The clinical, laboratory, genetic and molecular characteristics
of these three defects of TH action are the subject of this review. The first
defect, recognized 45years ago, produces resistance to TH and carries the
acronym, RTH. In the majority of cases it is caused by TH receptor β gene
mutations. It has been found in over 3000 individuals belonging to approximately
1000 families. Two relatively novel syndromes presenting reduced sensitivity to
TH involve membrane transport and metabolism of TH. One of them, caused by
mutations in the TH cell-membrane transporter MCT8, produces severe psychomotor
defects. It has been identified in more than 170 males from 90 families. A
defect of the intracellular metabolism of TH in 10 individuals from 8 families
is caused by mutations in the SECISBP2 gene required for the synthesis of
selenoproteins, including TH deiodinases.
MAJOR CONCLUSIONS: Defects at different steps along the pathway leading to TH
action at cellular level can manifest as reduced sensitivity to TH.
GENERAL SIGNIFICANCE: Knowledge of the molecular mechanisms involved in TH
action allows the recognition of the phenotypes caused by defects of TH action.
Once previously known defects have been ruled out, new molecular defects could
be sought, thus opening the avenue for novel insights in thyroid physiology.
This article is part of a Special Issue entitled Thyroid hormone signaling. The sodium/proton (Na/H) exchanger, Na,K-ATPase, and Ca2+-ATPase are membrane
ion pumps whose basal activities may be regulated by local nongenomic actions of
thyroid hormone and hormone analogues via a hormone receptor on plasma membrane
integrin αvβ3. System A amino acid transport and the activity of P-glycoprotein
(P-gp; ABCB1), a multidrug efflux pump, are also modulated by thyroid hormone
and αvβ3. Where signal transduction has been studied, the presence of the
hormone at the receptor is transduced by mitogen-activated protein kinase (MAPK)
isoforms (ERK1/2; p38) or phosphatidylinositol 3-kinase into local actions. The
existence of the cell surface receptor offers opportunities to pharmacologically
modify actions of these important transport functions with oparticulate
formulations of T4 and T3 that do not enter the cell. Such formulations may
reverse complex intracellular accumulations of H+, Na+, and Ca2+ that occur in
clinical settings such as ischemia. In addition, oparticulate
tetraiodothyroacetic acid (tetrac), a thyroid hormone analogue that inhibits
binding of T4 and T3 to integrin αvβ3 as well as certain other functions of the
integrin, may reverse P-gp-dependent resistance to anti-cancer drugs in tumor
cells. BACKGROUND: The secretory activity of Sertoli cells (SC) is dependent on ion
channel functions and protein synthesis and is critical to ongoing
spermatogenesis. The aim of this study was to investigate the mechanism of
action associated with a non-metabolizable amino acid [14C]-MeAIB
(alpha-(methyl-amino)isobutyric acid) accumulation stimulated by T4 and the role
of the integrin receptor in this event, and also to clarify whether the T4
effect on MeAIB accumulation and on Ca2+ influx culminates in cell secretion.
METHODS: We have studied the rapid and plasma membrane initiated effects of T4
by using 45Ca2+ uptake and [45C]-MeAIB accumulation assays, respectively.
Thymidine incorporation into DNA was used to monitor nuclear activity and
quinacrine to analyze the secretory activity on SC.
RESULTS: The stimulation of MeAIB accumulation byT4 appears to be mediated by
the integrin receptor in the plasma membrane since tetrac and RGD peptide were
able to nullify the effect of this hormone. In addition, T4 increases
extracellular Ca2+ uptake and Ca2+ from intracellular stocks to enhance nuclear
activity, but this genomic action seems not to influence SC secretion mediated
by T4. Also, the cytoskeleton and CIC-3 chloride channel contribute to the
membrane-associated responses of SC.
CONCLUSIONS: T4 integrin receptor activation ultimately determines the plasma
membrane responses on amino acid transport in SC, but it is not involved in
calcium influx, cell secretion or the nuclear effect of the hormone.
GENERAL SIGNIFICANCE: The integrin receptor activation by T4 may take a role in
plasma membrane processes involved in the male reproductive system. This paper and the following one (see the next issue of Acta Pharmaceutica
Hungarica) survey the biological roles and the related site-specific
physico-chemical parameters (basicity and lipophilicity) of the presently known
thyroid hormones (thyroxine, liothyronine and reverse liothyronine) and their
biological precursors (monoiodotyrosine and diiodotyrosine). Here the literature
of the thyroid hormone biochemistry, biosynthesis, plasma- and membrane
transport is summarized, focusing on the pH-dependent processes. Biosyntheses of
the thyroid hormones take place by oxidative coupling of two iodotyrosine
residues catalyzed by thyreoperoxidase in thyreoglobulin. The protonation state
of the precursors, especially that of the phenolic OH is crucial for the
biosynthesis, since anionic iodotyrosine residues can only be coupled in the
thyroid hormone biosyntheses. In the blood more than 99% of the circulating
thyroid hormone is bound to plasma proteins among which the thyroxine-binding
globulin and transthyretin are crucial. The amphiphilic character of the
hormones is assumed to be the reason why their membrane transport is an
energy-dependent, transport-mediated process, in which the organic anion
transporter family, mainly OATP1C1, and the amino acid transporters, such as
MCT8 play important roles. Liothyronine is the biologically active hormone; it
binds the thyroid hormone receptor, a type of nuclear receptor. There are two
major thyroid hormone receptor (TR) isoforms, alfa (TRalpha) and beta (TRbeta).
The activation of the TRalpha is associated with modifications in cardiac
behavior, while activation of the TRbeta is associated with increasing metabolic
rates, resulting in weight loss and reduction of blood plasma lipid levels. The
affinity of the thyroid hormones for different proteins depends on the
ionization state of the ligands. The site-specific physico-chemical
characterization of the thyroid hormones is of fundamental importance to
understand their (patho)physiological behavior and also, to influence their
therapeutic properties at the molecular level. Integrins are heterodimeric structural components of the plasma membrane whose
ligands include a large number of extracellular matrix (ECM) proteins. The
ligands contain Arg-Gly-Asp (RGD) sequences that enable recognition of ECM
proteins by as many as eight integrins, but other distinguishing features of the
proteins permit the integrins to generate intracellular signals specific to the
ECM molecules. Recently, integrin αvβ3 has been shown to have a panel of
previously unappreciated small molecule receptor sites for thyroid hormone and
hormone analogues, for dihydrotestosterone, and for resveratrol, a polyphenol
that has certain estrogen-like features. These binding sites are close to the
RGD recognition site of αvβ3. The thyroid hormone receptor site on the
extracellular domain of αvβ3 contains two domains with discrete functions in
terms of intracellular protein trafficking and gene expression. Occupancy of the
receptor by a deaminated thyroid hormone analogue, tetraiodothyroacetic acid
(tetrac), prevents cell responses to agonist thyroid hormones (L-thyroxine; 3,
5, 3'-triiodo-L-thyronine) and modulates expression of a number of cancer cell
survival pathway genes in an up- or downregulation pattern coherent to induction
of cell death. The small molecule thyroid hormone receptor on the integrin also
regulates activity of five vascular growth factor receptors and/or their
ligands, providing control of angiogenesis via specific pharmacologic regulation
of this thyroid hormone receptor. The resveratrol receptor induces programmed
cancer cell death via p53, even when the latter has undergone specific
mutations. There is also evidence for the presence of several receptors on
integrin αvβ3 for authentic steroids, including a dihydrotestosterone site that
supports proliferation of breast cancer cells that lack nuclear androgen and
estrogen receptors. The existence of these small molecule hormone receptors on
an integrin with a remarkably complex functional profile defines novel
pharmacologic options via individual small molecule receptor manipulation for
control of cancer cell behavior. This refinement of up-down control at the level
of discrete receptors is not a function of the use of αvβ3 antibody or RGD
peptides that occlude regions of the integrin. |
Which disorder is rated by Palmini classification? | Palmini classification system is used for classification of focal cortical dysplasia. | Tuberous sclerosis complex (TSC) and severe cortical dysplasia (CD), or CD type
II according to Palmini classification, share histopathologic similarities,
specifically the presence of cytomegalic neurons and balloon cells. In this
study we examined the morphologic and electrophysiologic properties of cells in
cortical tissue samples from pediatric patients with TSC and CD type II who
underwent surgery for pharmacoresistant epilepsy. Normal-appearing pyramidal
neurons from TSC and CD type II cases had similar passive membrane properties.
However, the frequency of excitatory postsynaptic currents (EPSCs) was higher in
neurons from TSC compared to severe CD cases, particularly the frequency of
medium- and large-amplitude synaptic events. In addition, EPSCs rise and decay
times were slower in normal cells from TSC compared to severe CD cases. Balloon
cells were found more frequently in TSC cases, whereas cytomegalic pyramidal
neurons occurred more often in CD type II cases. Both cell types were similar
morphologically and electrophysiologically in TSC and severe CD. These results
suggest that even though the histopathology in TSC and severe CD is similar,
there are subtle differences in spontaneous synaptic activity and topographic
distribution of abnormal cells. These differences may contribute to variable
mechanisms of epileptogenesis in patients with TSC compared with CD type II. PURPOSE: The purpose of this study is to determine the sensibility of each
imaging tool in identifying focal cortical dysplasia (FCD) in children and
adolescents with epilepsy and to define the prognostic factors of pediatric and
adolescent epilepsy surgery.
METHODS: We identified 48 children with FCD who underwent resective surgery and
analyzed their preoperative data. The results of various anatomic and functional
neuroimaging studies were compared for accuracy in locating the lesion. We also
investigated clinical factors that affected the outcome of surgical treatment.
KEY FINDINGS: Brain magnetic resoce imaging (MRI) was able to localize FCD in
30 patients and fluorodeoxyglucose positron emission tomography (FDG-PET) and/or
subtraction ictal single photon emission computed tomography (SPECT)
coregistered with MRI provided additional information that helped to define the
lesion in 13 patients. When comparing the pathologic results between a mild
malformation of cortical development (MCD) and FCD type I and II, we noted a
strong tendency for patients with FCD to have MRI abnormalities (p = 0.005). In
addition, severe pathologic features (Palmini's classification, FCD type II) (p
= 0.025) showed significant correlation with a better surgical outcome. To
define the primary epileptogenic area, various interictal epileptiform
discharges and the results of multimodal neuroimaging studies were helpful, and
younger age at the time of operation could aid in more favorable surgical
outcomes (p = 0.048).
SIGNIFICANCE: Our study showed a significant relationship between pathologic
grade and the detectability of FCD by brain MRI. In addition, early surgery can
be justified by showing that advanced neuroimaging studies in children with FCD
and even with extensive epileptiform discharges have a higher rate of success. Focal cortical dysplasia is a malformation of cortical development, which is the
most common cause of medically refractory epilepsy in the pediatric population
and the second/third most common etiology of medically intractable seizures in
adults.Both genetic and acquired factors are involved in the pathogenesis of
cortical dysplasia. Numerous classifications of the complex structural
abnormalities of focal cortical dysplasia have been proposed - from Taylor et
al. in 1971 to the last modification of Palmini classification made by Blumcke
in 2011. In general, three types of cortical dysplasia are recognized.Type I
focal cortical dysplasia with mild symptomatic expression and late onset, is
more often seen in adults, with changes present in the temporal lobe.Clinical
symptoms are more severe in type II of cortical dysplasia usually seen in
children. In this type, more extensive changes occur outside the temporal lobe
with predilection for the frontal lobes.New type III is one of the above
dysplasias with associated another principal lesion as hippocampal sclerosis,
tumor, vascular malformation or acquired pathology during early life.Brain MRI
imaging shows abnormalities in the majority of type II dysplasias and in only
some of type I cortical dysplasias.THE MOST COMMON FINDINGS ON MRI IMAGING
INCLUDE: focal cortical thickening or thinning, areas of focal brain atrophy,
blurring of the gray-white junction, increased signal on T2- and FLAIR-weighted
images in the gray and subcortical white matter often tapering toward the
ventricle. On the basis of the MRI findings, it is possible to differentiate
between type I and type II cortical dysplasia. A complete resection of the
epileptogenic zone is required for seizure-free life. MRI imaging is very
helpful to identify those patients who are likely to benefit from surgical
treatment in a group of patients with drug-resistant epilepsy.However, in type I
cortical dysplasia, MR imaging is often normal, and also in both types the
lesion seen on MRI may be smaller than the seizure-generating region seen in the
EEG. The abnormalities may also involve vital for life brain parts, where
curative surgery will not be an option. Therefore, other diagnostic imaging
techniques such as FDG PET, MEG, DTI and intra-cranial EEG are widely used to
establish the diagnosis and to decide on management.With advances in both
genetics and neuroimaging, we may develop a better understanding of patients
with drug-resistant epilepsy, which will help us to provide more successful
pharmacological and/or surgical treatment in the future. PURPOSE: The new International League Against Epilepsy (ILAE) classification for
focal cortical dysplasia (FCD) differentiates between patients with isolated FCD
(type 1) and FCD with an associated hippocampal sclerosis (HS) (type 3a). In
contrast to the former FCD classification by Palmini, which considered only
histologic features, the novel ILAE classification also relies on magnetic
resoce imaging (MRI) findings and presumed pathogenesis. We investigated in a
cohort of 100 patients with exclusively temporal FCD if the new subdivision of
FCD is reflected in clinical characteristics.
METHODS: Thirty-one patients with FCD type 1 and 50 patients with FCD type 3a in
the temporal lobe were included. In all patients MRI and histology of the FCD
were available. Both patient groups were compared to 19 patients with temporal
FCD type 2 with clearly different histologic appearance.
KEY FINDINGS: Patients with FCD type 1 and type 3a presented with similar
clinical features in many respects. In univariate analyses, no statistically
significant differences were found as to age at epilepsy onset (p = 0.07) and
epilepsy surgery (p = 0.14), a normal appearing neocortical temporal lobe (p =
0.08) or diagnosis of FCD by visual inspection of MRI (p = 0.08), preoperative
seizure frequency (p = 0.06), and the predomice of an epigastric aura (p =
0.08). The postoperative outcome was nearly identical 1 year (p = 0.8) and 2 (p
= 0.8), 3 (p = 0.8), 5 (p = 0.7), and 8 (p = 1.0) years postoperatively. Only
febrile seizures (p = 0.025) and an aura (p = 0.03) were significantly more
frequently reported in patients with FCD type 3a. Similar results were obtained
from a multivariate logistic regression analysis. Patients with FCD type 2 were
more different: Compared to FCD type 3a, age at epilepsy surgery was
significantly lower (p = 0.004) and auras (p = 0.005) were significantly less
frequently reported. Epigastric auras (p = 0.04) and febrile seizures (p =
0.025) occurred significantly less frequently in patients with FCD type 2
without HS compared to FCD type 3a. The diagnosis of an FCD was significantly
more frequently made (p = 0.03) by visual inspection of the MRI compared to FCD
type 1.
SIGNIFICANCE: Clinical features did not allow to clear separation of temporal
FCD types 1 and 3a. Statistically significant differences were seen in a history
of febrile seizures and the occurrence of auras more common in FCD type 3a.
However, FCD type 2 in the same localization but with different histology
presented with further differences such as more frequent FCD diagnosis by visual
inspection of MRI, earlier operation, and less frequent epigastric auras. For dozens of years, a variety of pathological findings have been revealed
through previous observations on surgically resected lesions from patients with
intractable epilepsy, including excessive number of neurons in the molecular
layer of cortices, unexpected existence of white matter neurons, and persistent
columnar structure. These findings have sometimes been referred to as
microdysgenesis (MD) or mild malformation of cortical development (mMCD), which
is defined as microscopic abnormalities of brain formations with no
macroscopical and neuroradiological findings. Taylor et al. (1971) described
surgical cases with giant neurons and bizarre, grotesque cells as "focal
dysplasia of the cerebral cortices with epilepsy." Since 1997, such
malformations have subsequently been referred to as focal cortical dysplasia
(FCD), in Greenfield's Neuropathology. Since early 2000, the definition of FCD
has gradually been given a broader interpretation than the case described by
Taylor et al., as shown in Palmini's classification (2004) or the newest
classification (2011) proposed by the Neuropathology Task Force of the
International League Against Epilepsy (ILAE). The ILAE classification describes
3 types of disease: Type I, Type II, and Type III. Type I is equivalent to some,
but not all, pathological phenotypes of MD or mMCD. Type II is Taylor's FCD
alone. Type III, which was not included in Palmini's classification, merges
brain malformation and other pathological findings. However, the reproducibility
of pathological diagnosis by using the ILAE classification was low, except for
Type IIb. Hence, future studies are necessary to provide further reliable
criteria for the pathological diagnosis of epilepsy patients. PURPOSE: The study analyzes the occurrence of high frequency oscillations in
different types of focal cortical dysplasia in 22 patients with refractory
epilepsy. High frequency oscillations are biomarkers for epileptic tissue, but
it is unknown whether they can reflect increasingly dysplastic tissue changes as
well as epileptic disease activity.
METHODS: High frequency oscillations (80-450 Hz) were visually marked by two
independent reviewers in all channels of intracranial implanted grid, strips,
and depth electrodes in patients with focal cortical dysplasia and refractory
epilepsy. Rates of high frequency oscillations in patients with pathologically
confirmed focal cortical dysplasia of Palmini type 1a and b were compared with
those in type 2a and b.
KEY FINDINGS: Patients with focal cortical dysplasia type 2 had significantly
more seizures than those with type 1 (p < 0.001). Rates of high frequency
oscillations were significantly higher in patients with focal cortical dysplasia
type 2 versus type 1 (p < 0.001). In addition, it could be confirmed that rates
of high frequency oscillations were significantly higher in presumed
epileptogenic areas than outside (p < 0.001).
SIGNIFICANCE: Activity of high frequency oscillations mirrors the higher
epileptogenicity of focal cortical dysplasia type 2 lesions compared to type 1
lesions. Therefore, rates of high frequency oscillations can reflect disease
activity of a lesion. This has implications for the use of high frequency
oscillations as biomarkers for epileptogenic areas, because a detailed analysis
of their rates may be necessary to use high frequency oscillations as a
predictive tool in epilepsy surgery. BACKGROUND AND PURPOSE: Malformation of cortical development (MCD) is a
well-known cause of drug-resistant epilepsy and focal cortical dysplasia (FCD)
is the most common neuropathological finding in surgical specimens from
drug-resistant epilepsy patients. Palmini's classification proposed in 2004 is
now widely used to categorize FCD. Recently, however, Blumcke et al. recommended
a new system for classifying FCD in 2011.
METHODS: We applied the new classification system in practical diagnosis of a
sample of 117 patients who underwent neurosurgical operations due to
drug-resistant epilepsy at Severance Hospital in Seoul, Korea.
RESULTS: Among 117 cases, a total of 16 cases were shifted to other FCD subtypes
under the new classification system. Five cases were reclassified to type IIIa
and five cases were categorized as dual pathology. The other six cases were
changed within the type I category.
CONCLUSIONS: The most remarkable changes in the new classification system are
the advent of dual pathology and FCD type III. Thus, it will be very important
for pathologists and clinicians to discriminate between these new categories.
More large-scale research needs to be conducted to elucidate the clinical
influence of the alterations within the classification of type I disease.
Although the new FCD classification system has several advantages compared to
the former, the correlation with clinical characteristics is not yet clear. To determine long-term efficacy and safety of epilepsy surgery in children and
adolescents with malformations of cortical development (MCD) and to identify
differences in seizure outcome of the various MCD subgroups. Special focus was
set on the newly introduced International League Against Epilepsy (ILAE)
classification of focal cortical dysplasia (FCD).
STUDY DESIGN: This is a single center retrospective cross-sectional analysis of
prospectively collected data.
INCLUSION CRITERIA: age at surgery <18 years, pre-surgical evaluation and
epilepsy surgery performed at the Vienna pediatric epilepsy center,
histologically proven MCD, complete follow-up data for at least 12 months.
Clinical variables evaluated: type and localization of MCD, type of surgery and
a variety of clinical characteristics reported to be associated with
(un-)favorable outcomes. MCD were classified following the existing
classification schemes (Barkovich et al., 2012. Brain. 135, 1348-1369; Palmini
et al., 2004. Neurology. 62, S2-S8) and the ILAE classification for FCD recently
proposed by Blümcke in 2011. Seizure outcome was classified using the ILAE
classification proposed by Wieser in 2001.
RESULTS: 60 Patients (51.7% male) were included. Follow up was up to 14 (mean
4.4 ± 3.2) years. Mean age at surgery was 8.0 ± 6.0 (median 6.0) years; mean age
at epilepsy onset was 2.9 ± 3.2 (median 2.0) years; duration of epilepsy before
surgery was 4.8 ± 4.4 (median 3.0) years. 80% of the patients were seizure free
at last follow-up. AEDs were successfully withdrawn in 56.7% of all patients.
Extended surgery, lesion localization in the temporal lobes and absence of
inter-ictal spikes in postsurgical EEG recordings were predictive of favorable
seizure outcomes after surgery. However, no association was found between
outcome and MCD sub-types. Epilepsy surgery is highly effective in carefully
selected drug-resistant children with MCD. Surrogate markers for complete
resection of the epileptogenic zone remain the only significant predictors for
seizure freedom after surgery. Meningioangiomatosis is a rare, benign, developmental, or hamartomatous lesion
which may involve the leptomeninges and underlying brain parenchyma.
Histologically, meningioangiomatosis is marked by a proliferation of blood
vessels in the parenchyma, rimmed by collars of spindled meningothelial cells.
There are anecdotal reports of an association of meningioangiomatosis with focal
cortical dysplasia. We retrospectively analyzed the clinical, histopathologic,
and treatment outcomes of 16 patients with a diagnosis of meningioangiomatosis,
specifically investigating these cases for evidence of adjacent focal cortical
dysplasia. Patients ranged in age from 1 to 34 years (median 18), 12 of whom had
medically-intractable epilepsy as their presenting symptom. No patients in this
study had a confirmed diagnosis of neurofibromatosis type II. Four patients
(25%) were found to have fibrous meningiomas associated with the
meningioangiomatosis. Ten of the 12 patients (83%) who had adequate tissue
excised adjacent to the meningioangiomatosis demonstrated evidence of focal
cortical dysplasia, with 6 of those (60%) classified as Palmini type IA, and 4
patients (40%) classified as Palmini type IIA. Seven of the patients (44%) had
no post-operative seizures, and were off anti-epileptic drugs, while 2 patients
relapsed, and required pharmacologic treatment for seizure control. This study
therefore presents evidence to support inclusion of meningioangiomatosis as a
focal cortical dysplasia-associated entity, as suggested by the ILAE
classification (type IIIc). As focal cortical dysplasia is a developmental
malformation, its association with meningioangiomatosis supports a developmental
etiology of sporadic meningioangiomatosis. |
what is the role of erythropoietin in cardiac regeneration after myocardial infarction? | In preclinical studies, erythropoietin improved cardiac function and perfusion by angiomyogenesis and protection of cardiomyocytes in myocardial infarction indicating that erythropoietin may play a role in the stimulation of cell regeneration under normal physiologic conditions and in patients with myocardial injury. Epo overexpression was found to enhance the cellular regenerative properties of MSCs by both autocrine and paracrine pathways. However, results from recent clinical trials did not support beneficial effects of cytokine therapy with erythropoietin in patients with myocardial infarction. | OBJECTIVES: We assessed the effects of erythropoietin (EPO) treatment in a rat
model of post-myocardial infarction (MI) heart failure.
BACKGROUND: Erythropoietin, traditionally known as a hematopoietic hormone, has
been linked to neovascularization. Whereas administration of EPO acutely after
MI reduces infarct size and improves cardiac function, its role in the failing
heart is unknown.
METHODS: Rats underwent coronary ligation or sham surgery. Rats with MI were
randomly assigned to: untreated (MI), a single bolus of EPO immediately after MI
induction (MI-EPO-early), EPO treatment immediately after MI and once every
three weeks (MI-EPO-early+late), and EPO treatment starting three weeks after
induction of MI, once every three weeks (MI-EPO-late). After nine weeks,
hemodynamics, infarct size, myosin heavy chain (MHC) isoforms, myocyte
hypertrophy, and capillary density were measured.
RESULTS: Erythropoietin treatment started immediately after MI (MI-EPO-early and
MI-EPO-early+late) resulted in a 23% to 30% reduction in infarct size (p < 0.01)
and, accordingly, hemodynamic improvement. Erythropoietin treatment, started
three weeks after MI (MI-EPO-late), did not affect infarct size, but resulted in
an improved cardiac performance, reflected by a 34% reduction in left
ventricular end-diastolic pressure (p < 0.01), and 46% decrease in atrial
natriuretic peptide levels (p < 0.05). The improved cardiac function was
accompanied by an increased capillary density (p < 0.01), an increased
capillary-to-myocyte ratio (p < 0.05), and a partial reversal of beta-MHC (p <
0.05) in all treated groups.
CONCLUSIONS: In addition to its effect on infarct size reduction, EPO treatment
improves cardiac function in a rat model of post-MI heart failure. This
observation may be explained by neovascularization, associated with an increased
alpha-MHC expression. Erythropoietin (EPO) has been suggested to have a cardioprotective effect
against ischemia. The purpose of this study was to examine the effects of EPO on
cardiac remodeling after myocardial infarction (MI). MI was induced by ligation
of the coronary artery in Wistar rats. The rats with MI were randomly divided
into untreated MI and two EPO-treated MI groups. EPO was administered
subcutaneously by injection once a day for 4 days after MI at 5000 U/kg or 3
times a week for 4 weeks at 1000 U/kg. Five days after MI, EPO prevented the
increase in activated caspase 3, matrix metalloproteinase-2, and transcriptional
activation of activator protein-1 in non-infarcted myocardium. Four weeks after
MI, left ventricular weight, left ventricular end-diastolic pressure, and left
ventricular dimension were increased, and ejection fraction and E wave
deceleration time were decreased. EPO significantly attenuated this ventricular
remodeling and systolic and diastolic dysfunction. In addition, EPO
significantly attenuated the interstitial fibrosis and remodeling-related gene
expression in non-infarcted myocardium. Furthermore, EPO significantly enhanced
angiogenesis and reduced apoptotic cell death in peri-infarcted myocardium. In
conclusion, when administered after MI, EPO prevents cardiac remodeling and
improves ventricular function with enhanced angiogenesis and reduced apoptosis. OBJECTIVES: We investigated the effects of erythropoietin (EPO) on
neovascularization and cardiac function after myocardial infarction (MI).
BACKGROUND: Erythropoietin exerts antiapoptotic effects and mobilizes
endothelial progenitor cells (EPCs).
METHODS: We intravenously administered EPO (1,000 IU/kg) immediately [EPO(0)
group], 6 h [EPO(6h) group], or 1 week [EPO(1wk) group] after the permanent
ligation of the coronary artery in dogs. Control animals received saline
immediately after the ligation.
RESULTS: The infarct size 6 h after MI was significantly smaller in the EPO(0)
group than in the control group (61.5 +/- 6.0% vs. 22.9 +/- 2.2%). One week
after MI, the circulating CD34-positive mononuclear cell numbers in both the
EPO(0) and the EPO(6h) groups were significantly higher than in the control
group. In the ischemic region, the capillary density and myocardial blood flow 4
weeks after MI was significantly higher in both the EPO(0) and the EPO(6h)
groups than in the control group. Four weeks after MI, left ventricular (LV)
ejection fraction in the EPO(6h) (48.6 +/- 1.9%) group was significantly higher
than that in either the control (41.9 +/- 0.9%) or the EPO(1wk) (42.6 +/- 1.2%)
group but significantly lower than that in the EPO(0) group (56.1 +/- 2.3%). The
LV end-diastolic pressure 4 weeks after MI in both the EPO(0) and the EPO(6h)
groups was significantly lower than either the control or the EPO(1wk) group.
Hematologic parameters did not differ among the groups.
CONCLUSIONS: In addition to its acute infarct size-limiting effect, EPO enhances
neovascularization, likely via EPC mobilization, and improves cardiac
dysfunction in the chronic phase, although it has time-window limitations. BACKGROUND: Transplantation of marrow stromal cells (MSC) has been shown to
improve heart perfusion and cardiac function after ischemia. Erythropoietin
(EPO) is capable of inducing angiogenesis and inhibiting cell apoptosis. The aim
of this study was to investigate the effect of EPO on the therapeutic potency of
MSC transplantation in a rat model of myocardial infarction.
METHODS: MSC viability was detected by MTT and flow cytometry following culture
in serum-free medium for 24 h with or without EPO. Release of vascular
endothelial growth factor (VEGF) by MSC incubated with different doses of EPO
was assayed using ELISA. Immediately after coronary ligation, autologous MSC (3
x 10(6) cells) were injected into the ischemic myocardium (MSC and MSC-EPO
groups). EPO (3,000 U/kg body weight) was injected daily for 3 consecutive days
starting 1 day prior to ligation. The same EPO dose was also injected for
consecutive 3 days starting 15 days after surgery (EPO and MSC-EPO groups).
Control animals were injected saline solution for the same time period. Cardiac
function was assessed by echocardiography 2 and 21 days after surgery,
respectively. Western blot and immunohistological assessments were performed to
examine the effects of treatments.
RESULTS: In vitro, EPO inhibited MSC apoptosis induced by serum-free medium and
increased vascular endothelial growth factor (VEGF) release by MSC. In vivo,
cardiac infarct size was significantly smaller, cardiac function significantly
improved, and capillary density obviously higher in the MSC and EPO groups than
in the control group. Combined treatment with EPO infusion and MSC
transplantation demonstrated a further decrease in infarct size, a further
improvement in cardiac function, and a further increase in capillary density
compared with MSC or EPO alone. Furthermore, a higher ratio of phosphorylated
Akt to total Akt was measured by Western blot; Bcl-2 was upregulated and Bax was
downregulated by immunohistochemistry in the MSC-EPO group compared to the other
three groups.
CONCLUSION: Transplantation of MSC combined with EPO infusion is superior to MSC
monotherapy for angiogenesis and cardiac function recovery. We postulated that combining erythropoietin (EPO) infusion with bone marrow
mesenchymal stem cells (MSC) delivery may give better prognosis in a rat
infarcted heart. Acute myocardial infarction (MI) model was developed by
coronary artery ligation. Animals were grouped (n=18) to receive intramyocardial
injection of 30 microl saline solution without cells (EPO and control groups) or
with 3x10(6) MSC from transgenic green fluorescent protein (GFP)+ male mice (MSC
and MSC-EPO groups). The animals received either 5000 U/kg body weight EPO (EPO
and MSC-EPO groups) or saline solution (MSC and control groups) for 7 days after
MI. Cardiac functions were measured by echocardiography and cardiac tissue was
harvested for immunohistological studies 3 weeks after surgery. We observed
regeneration of MSC in and around the infarcted myocardium in MSC and MSC-EPO
groups. Capillary density was markedly enhanced with significantly smaller
infarct size and reduced fibrotic area in MSC-EPO group as compared with other
three groups. A smaller left ventricular (LV) diastolic dimension and a higher
LV fractional shortening were observed in MSC-EPO group than in other three
groups. Transplantation of MSC combined with cytokine EPO is superior to either
of the monotherapy approach for angiomyogenesis and cardiac function recovery. AIMS: Erythropoietin (EPO) improves cardiac function and induces
neovascularization in chronic heart failure (CHF), although the exact mechanism
has not been elucidated. We studied the effects of EPO on homing and
incorporation of endothelial progenitor cells (EPC) into the myocardial
microvasculature and myocardial expression of angiogenic factors.
METHODS AND RESULTS: CHF was induced in rats by coronary artery ligation
resulting in myocardial infarction (MI) after bone marrow had been replaced by
human placental alkaline phosphatase (hPAP) transgenic cells. We studied the
effects of darbepoetin alfa treatment (EPO, 40 microg/kg, every 3 weeks,
starting 3 weeks after MI) on longitudinal changes in left ventricular (LV)
function, circulating EPC, myocardial histology, and expression of vascular
endothelial growth factor (VEGF) determined 9 weeks after MI. EPO prevented
LV-dilatation and improved cardiac function (all P < 0.05), which was associated
with 42% increased capillary growth (P < 0.01). EPO-induced mobilization of EPC
from the bone marrow (P < 0.01), which resulted in a three-fold increased homing
of EPC into the cardiac microvasculature. The percentage of the endothelium that
consisted of bone marrow derived cells was significantly increased (3.9 +/- 0.5
vs. 11.4 +/- 1%, P < 0.001) comprising 30% of the newly formed capillaries. In
addition, EPO treatment resulted in a 4.5-fold increased myocardial expression
of VEGF, which correlated strongly with neovascularization (r = 0.67; P <
0.001). VEGF was equally expressed by endothelial cells of myocardial and bone
marrow origin.
CONCLUSION: EPO-induced neovascularization in post-MI heart failure is mediated
through a combination of EPC recruitment from the bone marrow and increased
myocardial expression of VEGF. AIMS: Mesenchymal stromal cells (MSCs) possess intrinsic features that identify
them as useful for treating ischaemic syndromes. Poor in vivo
survival/engraftment of MSCs, however, limits their overall effectiveness. In
this work, we tested whether genetically engineering MSCs to secrete
erythropoietin (Epo) could represent a better therapeutic platform than MSCs in
their native form.
METHODS AND RESULTS: MSCs from C57Bl/6 mice were retrovirally transduced with
either an empty vector or one that causes the production of Epo and were then
analysed for the alterations in angiogenic and survival potential. Using a mouse
model of myocardial infarction (MI), the regenerative potential of null MSCs and
Epo-overexpressing MSCs (Epo+MSCs) was assessed using serial echocardiogram and
invasive haemodynamic measurements. Infarct size, capillary density and
neutrophil influx were assessed using histologic techniques. Using in vitro
assays coupled with an in vivo Matrigel plug assay, we demonstrate that
engineering MSCs to express Epo does not alter their immunophenotype or
plasticity. However, relative to mock-modified MSCs [wild-type (WT)-MSCs],
Epo+MSCs are more resilient to apoptotic stimuli and initiate a more robust
host-derived angiogenic response. We also identify and characterize the
autocrine loop established on MSCs by having them secrete Epo. Furthermore, in a
murine model of MI, animals receiving intracardiac injections of Epo+MSCs
exhibited significantly enhanced cardiac function compared with WT-MSCs and
saline-injected control animals post-MI, owing to the increased myocardial
capillary density and the reduced neutrophilia.
CONCLUSION: Epo overexpression enhances the cellular regenerative properties of
MSCs by both autocrine and paracrine pathways. Granulocyte colony-stimulating factor (G-CSF) and erythropoietin are two
cytokines that have been demonstrated to improve cardiac function and perfusion
in myocardial infarction. G-CSF was initially evaluated as a stem cell mobilizer
and erythropoietin as a cytoprotective agent. However, both cytokines have
direct cytoprotective effects and stem cell-mobilizing ability. Direct
cytoprotective effects of both cytokines are commonly mediated by the Jak-STAT
pathway. In preclinical study, G-CSF and erythropoietin improved cardiac
function and perfusion by angiomyogenesis and protection of cardiomyocytes in
myocardial infarction. However, results from recent clinical trials did not
support beneficial effects of cytokine therapy with G-CSF or erythropoietin
alone in patients with myocardial infarction. Further studies are required to
elucidate the mechanism of action and to improve therapeutic efficacy by
employing novel strategies, such as combined cytokines. Mobilization of bone marrow-derived stem cells (BMCs) was shown to have
protective effects after myocardial infarction (MI). However, the classical
mobilizing agent, granulocyte-colony stimulating factor (G-CSF) relapsed after
revealing an impaired homing capacity. In the search for superior cytokines,
erythropoietin (EPO) appears to be a promising agent. Therefore, we analyzed in
a murine model of surgically induced MI the influence of EPO treatment on
survival and functional parameters as well as BMC mobilization, homing, and
effect on resident cardiac stem cells (CSCs). Human EPO was injected
intraperitoneally after ligation of the left anterior descendens (LAD) for 3
days with a total dose of 5000 IU/kg 6 and 30 days after MI, and pressure volume
relationships were investigated in vivo. Cardiac tissues were analyzed by
histology. To show the effect on BMCs and CSCs, FACS analyses were performed.
Homing factors were analyzed by quantitative reverse transcription-polymerase
chain reaction (qRT-PCR) and ELISA. EPO-treated animals showed a significant
improvement of survival post-MI (62 vs. 36%). At days 6 and 30, all hemodynamic
parameters associated with attenuated remodeling, enhanced neovascularization,
and diminished apoptotic cells in the peri-infarct area were improved. BMC
subpopulations (CD31(+), c-kit(+), and Sca-1(+) cells) were mobilized, and
homing of Sca-1(+) and CXCR4(+) BMCs toward an SDF-1 gradient into the ischemic
myocardium was enhanced. However, there was no beneficial effect on CSCs. We
have shown that EPO application after MI shows cardioprotective effects. This
may be explained by mobilization of BMCs, which are homing via the CXCR-4/SDF-1
axis. However, EPO has no beneficial effects on resident CSCs. Therefore, new
treatment regimes using EPO together with other agents may combine complementary
beneficial effects preventing ischemic cardiomyopathy. The regenerative capacity of the myocardium and its blood vessels has now been
well demonstrated. The cytokines granulocyte colony-stimulating factor,
erythropoietin, and stem cell factor may play a role in helping to stimulate
cell regeneration under normal physiologic conditions and in patients with
myocardial injury. After an ischemic insult, cytokines are released into the
peripheral circulation and signal for the mobilization of stem cells. In
experimental cardiac injury models, the addition of cytokines has been shown to
improve myocardial function with and without the concurrent use of stem cell
therapy. Preliminary studies in humans using cytokine therapy alone for treating
myocardial infarction have been disappointing. Future studies in patients with
myocardial injury need to examine the use of various combinations of cytokines,
with and without the addition of intravascular stem cell infusions or direct
stem cell injections. Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes
beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO
injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI)
model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac
injection. Compared to myocardial infarction control group (MIC), EPO
significantly improved left ventricular function (n =11-14, P < 0.05) and
decreased right ventricular wall stress (n = 8, P < 0.05) assessed by
pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction
size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater
capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8,
P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial
apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P=
0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by
quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells
were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral
blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001).
Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in
MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial
functions following MI, which may attribute to the improved early recruitment of
c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant
SDF-1. BACKGROUND: Expanded endothelial progenitor cells (eEPC) improve global left
ventricular function in experimental myocardial infarction (MI). Erythropoietin
beta (EPO) applied together with eEPC may improve regional myocardial function
even further by anti-apoptotic and cardioprotective effects. Aim of this study
was to evaluate intramyocardial application of eEPCs and EPO as compared to
eEPCs or EPO alone in experimental MI.
METHODS AND RESULTS: In vitro experiments revealed that EPO dosed-dependently
decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA
expression in eEPC of proangiogenic and proinflammatory mediators measured by
TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion
of the left anterior descending coronary artery of nude rats (n = 8-9). After
myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel
density were enhanced in the border zone of the infarct area. Moreover,
apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to
transplantation of eEPCs alone. Regional wall motion of the left ventricle was
measured using Magnetic Resoce Imaging. After injection of eEPC in the
presence of EPO regional wall motion significantly improved as compared to
injection of eEPCs or EPO alone.
CONCLUSION: Intramyocardial transplantation of eEPC in the presence of EPO
during experimental MI improves regional wall motion. This was associated with
an increased local inflammation, vasculogenesis and survival of the transplanted
cells. Local application of EPO in addition to cell therapy may prove beneficial
in myocardial remodeling. Animal models that mimic cardiovascular diseases are indispensable tools for
understanding the mechanisms underlying the diseases at the cellular and
molecular level. This review focuses on various methods in preclinical research
to create small animal models of cardiac diseases, such as myocardial
infarction, dilated cardiomyopathy, heart failure, myocarditis and cardiac
hypertrophy, and the related stem cell treatment for these diseases. Erythropoietin improves myocardial function and enhances re-endothelialisation.
Aim of this study was to analyse progenitor cell mobilisation and restenosis in
patients from the Regeneration of Vital Myocardium in ST-Segment Elevation
Myocardial Infarction by Erythropoietin (REVIVAL-3) study. Patients with STEMI
undergoing percutaneous coronary intervention (PCI) were randomly assigned to
Epoetin beta (EPO) (n=68) or placebo (n=70). Drug-eluting stents (DES) were
utilised in 93% of patients receiving EPO and in 95% of patients receiving
placebo (p=0.83). Serial venous blood samples were drawn; CD133+ progenitor
cells were quantified by four-colour flow cytometry and cytokines interleukin
(IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumour necrosis factor (TNF) alpha were
analysed by cytometric bead array. Forty-eight hours after PCI a significant
increase in CD133+ progenitor cells was observed in the EPO group. Yet, no
differences in plasma cytokines were found. Quantitative coronary angiography
after six months revealed an increase in segment diameter stenosis in the EPO
group (32 ± 19% vs. 26 ± 14%, p=0.046). However, this increase in neointima
generation was not associated with progenitor cell mobilisation. EPO in patients
with STEMI treated with PCI is associated with an increase in diameter stenosis
that is not associated with circulating progenitor cells. BACKGROUND: Endothelial progenitor cells (EPCs) participate in vascular repair
and angiogenesis. Thus, EPC transplantation into ischemic myocardium might
improve cardiac function; however, the vast majority of cells die within a short
period. The present study was designed to investigate whether exogenous
erythropoietin (EPO) delivery could improve the survival of transplanted EPCs
and enhance the efficiency of EPC-based cell therapy.
METHODS: Myocardial infarction was induced in wild-type mice by ligating the
left anterior descending coronary artery. Enhanced green fluorescent
protein-EPCs with or without EPO were transplanted into peri-infarct myocardium.
Enhanced green fluorescent protein-EPCs were detected 7 and 28 d after surgery.
The amount of circulating EPCs was analyzed 3 and 28 d after surgery. The
stromal cell-derived factor-1α and vascular endothelial growth factor
concentrations, microvessel density, apoptosis, fibrosis in the peri-infarct
myocardium, and cardiac function were compared among the groups.
RESULTS: More enhanced green fluorescent protein-EPCs were found in the hearts
treated with EPC + EPO than in those treated with EPC alone. The circulating EPC
level was markedly elevated after EPC + EPO treatment compared with EPC
application alone. Stromal cell-derived factor-1α and vascular endothelial
growth factor were increased accordingly, along with increased microvessel
density, decreased apoptosis, and reduced fibrosis in the peri-infarct
myocardium. Left ventricular fractional shortening was greater and the
interventricular septum was thicker after EPC + EPO treatment compared with EPC
treatment alone.
CONCLUSIONS: EPO improved the efficiency of EPC therapy in mice with myocardial
infarction. This effect was associated with enhanced transplanted EPC survival
and autologous EPC mobilization. BACKGROUND: In experimental models of acute myocardial infarction (AMI),
erythropoietin (EPO) reduces infarct size and improves left ventricular (LV)
function. However, in the clinical setting, the effect of EPO in AMI was
unclear. We conducted a systematic review and meta-analysis of randomized
controlled trials (RCTs) of EPO to explore the safety and therapeutic effects of
EPO in patients with AMI.
METHODS: We identified reports of RCTs comparing EPO to placebo for AMI in adult
humans in PubMed, Cochrane Central Register of Controlled Trials, and EMBASE.
Outcomes included all-cause mortality, major cardiovascular events, cardiac
function by LV ejection fraction and infarct size.
RESULTS: We included 13 articles of RCTs with data for 1,564 patients.
Erythropoietin therapy did not improve LV ejection fraction (weighted mean
difference [WMD] 0.33, 95% CI -1.90 to 1.24, P = .68) and had no effect on
infarct size, as measured by cardiac magnetic resoce imaging (WMD -0.12,
-2.16 to 1.91, P = .90) or serum peak value of creatine kinase-MB (WMD -2.01,
-25.70 to 21.68, P = .87). Erythropoietin treatment did not decrease the risk of
total adverse cardiac events (relative risk [RR] 1.02, 0.65-1.61, P = .92).
Erythropoietin treatment also failed to decrease the risk of heart failure (RR,
0.69, 0.27-1.72, P = .42) and all-cause mortality (RR 0.55, 0.22-1.33, P = .18).
Moreover, EPO had no effect on the risk of stent thrombosis (RR, 0.69,
0.29-1.64, P = .40).
CONCLUSION: Erythropoietin in patients with AMI seems to have no clinical
benefit for heart function or reducing infarct size, cardiovascular events, and
all-cause mortality. Erythropoietin may not be a choice for patients with AMI. |
Does Rad9 interact with Aft1 in S.cerevisiae? | Yes. Rad9 functions together with Aft1 on DNA damage-prone chromatin to facilitate genome surveillance, thereby ensuring rapid and effective response to possible DNA damage events. | DNA damage response and repair proteins are centrally involved in genome
maintece pathways. Yet, little is known about their functional role under
non-DNA damage-inducing conditions. Here we show that Rad9 checkpoint protein,
known to mediate the damage signal from upstream to downstream essential
kinases, interacts with Aft1 transcription factor in the budding yeast. Aft1
regulates iron homeostasis and is also involved in genome integrity having
additional iron-independent functions. Using genome-wide expression and
chromatin immunoprecipitation approaches, we found Rad9 to be recruited to 16%
of the yeast genes, often related to cellular growth and metabolism, while
affecting the transcription of ∼2% of the coding genome in the absence of
exogenously induced DNA damage. Importantly, Rad9 is recruited to fragile
genomic regions (transcriptionally active, GC rich, centromeres, meiotic
recombination hotspots and retrotransposons) non-randomly and in an
Aft1-dependent manner. Further analyses revealed substantial genome-wide
parallels between Rad9 binding patterns to the genome and major activating
histone marks, such as H3K36me, H3K79me and H3K4me. Thus, our findings suggest
that Rad9 functions together with Aft1 on DNA damage-prone chromatin to
facilitate genome surveillance, thereby ensuring rapid and effective response to
possible DNA damage events. |
Are there any desmins present in plants? | No. Desmins are type III intermediate filament (IF) proteins that have been identified to date only in metazoa (human, Danio rerio, bovine). Desmins are also associated with severe forms of skeletal, cardiac and myofibrillar myopathies. | We investigated the marker profile of human ascitic and cultured mesothelial
cells, and compared it to that of ovarian carcinoma cells which are related in
terms of their histogenesis, unrelated colon carcinomas being used as reference.
Mesothelial and ovarian carcinoma cells could not be distinguished by
(intermediate) filament typing, using monoclonal antibodies (MAbs) to keratins,
vimentins and desmins. Colon carcinomas differed from mesothelial cells and
ovarian carcinomas by the absence of keratin-7 filaments. The epithelial marker
BW 495/36 was completely negative on mesothelial cells and positive on all
ovarian and colon carcinoma cells. While CEA was found on about 85% of all colon
carcinomas, CEA expression on mesothelial cells and ovarian carcinoma cells was
below 20%. The ovarian carcinoma markers (OV-TL 3, OV-TL 10, OC 125, MOV 18)
were strongly positive on ovarian carcinomas and negative on colon carcinomas
(or limited to traces of immunofluorescence on some samples). Although the
mesothelial cells showed weak or negative reactivity with these markers, OC 125
antigen was found by immunoelectron microscopy on the surface of cultured
mesothelial cells, and was shed in the culture supernatant at concentrations of
50, 28, and 25 CA 125 U/ml/10(4) positive cells. This suggests that mesothelial
cells may be responsible for the synthesis of CA 125 in ascitic fluid. The data
indicate that ovarian carcinomas, mesothelial cells and colon carcinomas can be
distinguished using a combination of anti-keratin antibodies with BW 495/36 and
anti-ovarian carcinoma markers. Desmin is a muscle-specific protein and a constitutive subunit of the
intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an
early marker of skeletal muscle myogenesis. We have characterized a clone of
desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The
full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino
acids. The predicted amino acid sequence of the zebrafish desmin shares a high
degree of similarity to other vertebrate desmins, but also contains a sequence
at the carboxyl terminal of the tail domain that is unique to the zebrafish. It
carries many features which are distinctive of IF subunit proteins. These
include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament
signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the
carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the
3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single
polyadenylation site. The temporal and spatial expression patterns of desmin
mRNA during early zebrafish development were studied. The onset of desmin
expression occurred at the 1-3 somite stage (11 hpf). It increased throughout
somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and
decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA
was initially localised exclusively to the somites, but was subsequently also
detected in other musculature in the developing heart and fins. The onset of
expression and the spatial localization of desmin mRNA in the zebrafish
coincides with that reported for MyoD and myogenin. Mutations in desmin have been associated with a subset of human myopathies.
Symptoms typically appear in the second to third decades of life, but in the
most severe cases can manifest themselves earlier. How desmin mutations lead to
aberrant muscle function, however, remains poorly defined. We created a series
of four mutations in rat desmin and tested their in vitro filament assembly
properties. RDM-G, a chimera between desmin and green fluorescent protein,
formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro
assembly at the unit-length filament stage, while RDM-3 had more subtle effects
on assembly. When expressed in cultured rat neonatal cardiac myocytes via
adenovirus infection, these mutant proteins disrupted the endogenous desmin
filament to an extent that correlated with their defects in in vitro assembly
properties. Disruption of the desmin network by RDM-1 was also associated with
disruption of plectin, myosin, and alpha-actinin organization in a significant
percentage of infected cells. In contrast, expression of RDM-2, which is similar
to previously characterized human mutant desmins, took longer to disrupt desmin
and plectin organization and had no significant effect on myosin or
alpha-actinin organization over the 5-day time course of our studies. RDM-3 had
the mildest effect on in vitro assembly and no discernable effect on either
desmin, plectin, myosin, or alpha-actinin organization in vivo. These results
indicate that mutations in desmin have both direct and indirect effects on the
cytoarchitecture of cardiac myocytes. We describe a revised and expanded database on human intermediate filament
proteins, a major component of the eukaryotic cytoskeleton. The family of 70
intermediate filament genes (including those encoding keratins, desmins, and
lamins) is now known to be associated with a wide range of diverse diseases, at
least 72 distinct human pathologies, including skin blistering, muscular
dystrophy, cardiomyopathy, premature aging syndromes, neurodegenerative
disorders, and cataract. To date, the database catalogs 1,274 manually-curated
pathogenic sequence variants and 170 allelic variants in intermediate filament
genes from over 459 peer-reviewed research articles. Unrelated cases were
collected from all of the six sequence homology groups and the sequence
variations were described at cDNA and protein levels with links to the related
diseases and reference articles. The mutations and polymorphisms are presented
in parallel with data on protein structure, gene, and chromosomal location and
basic information on associated diseases. Detailed statistics relating to the
variants records in the database are displayed by homology group, mutation type,
affected domain, associated diseases, and nucleic and amino acid substitutions.
Multiple sequence alignment algorithms can be run from queries to determine DNA
or protein sequence conservation. Literature sources can be interrogated within
the database and external links are provided to public databases. The database
is freely and publicly accessible online at www.interfil.org (last accessed 13
September 2007). Users can query the database by various keywords and the search
results can be downloaded. It is anticipated that the Human Intermediate
Filament Database (HIFD) will provide a useful resource to study human genome
variations for basic scientists, clinicians, and students alike. Mutations in the intermediate filament (IF) protein desmin cause severe forms of
myofibrillar myopathy characterized by partial aggregation of the
extrasarcomeric desmin cytoskeleton and structural disorganization of
myofibrils. In contrast to prior expectations, we showed that some of the known
disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are
assembly-competent and do allow formation of bona fide IFs in vitro and in vivo.
We also previously demonstrated that atomic force microscopy can be employed to
measure the tensile properties of single desmin IFs. Using the same approach on
filaments formed by the aforementioned mutant desmins, we now observed two
different omechanical behaviors: DesA360P exhibited tensile properties
similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited
local variations in their tensile properties along the filament length. Based on
these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle
disease by altering the specific biophysical properties of the desmin filaments,
thereby compromising both its mechanosensing and mechanotransduction ability. |
Are viruses involved in the etiology of human subacute thyroiditis? | Subacute thyroiditis (SAT) is an inflammatory disorder of the thyroid caused probably by viruses
The principal classes of viruses involed in SAT include Epstein Barr and Retroviridae | The etiology of subacute (de Quervain's) thyroiditis (SAT) is uncertain,
although it probably represents a nonspecific inflammatory response by the
thyroid to a variety of viruses. It has been suggested that nonimmune processes
are involved in SAT patients who have negative autoantibody titers. The disease
has a variable course; although it is self-limited in most cases, some patients
develop transient hypothyroidism, and others do not during the recovery period.
The present study was performed to evaluate the occurrence of TSH receptor
antibody (TRAb), measured by RRA (TSH binding inhibitor), TRAb measured by
stimulation assay (thyroid-stimulating antibody), and TRAb measured by blocking
assay [TSH-blocking antibody (TSH-BAb)] activity in 68 patients with SAT who had
negative autoantibody titers. The patients were divided into 2 groups: group I,
31 patients who developed hypothyroidism during the recovery period; and group
II, 37 patients who remained euthyroid during recovery. Positive immunoglobulin
activity occurred in about 20% of group I patients during follow-up, but in only
3% of group II patients. About 20% of group I patients developed positive
TSH-BAb activity and were hypothyroid, requiring exogenous hormone therapy for
1.2-3.5 yr, whereas hypothyroidism was relatively transient in group I patients
who had negative TSH-BAb activity (2-6 months). Although increased TSH-BAb
activity may account for hypothyroidism in some patients with SAT, the precise
mechanism for the development of transient hypothyroidism in SAT remains
enigmatic. 1. Am J Med. 1971 Jul;51(1):97-108. doi: 10.1016/0002-9343(71)90327-5. We present a 25-year-old woman with amyloid goiter due to hypersensitivity
vasculitis, who developed transient thyrotoxicosis resembling subacute
thyroiditis. She received prednisolone (20 mg/ day) for three years for
hypersensitivity vasculitis, and was diagnosed as having secondary amyloidosis
by biopsies of the stomach, rectum and kidneys. She noticed neck swelling with
severe right neck tenderness, palpitation, hyperhidrosis and weight loss. An
elastic firm diffuse goiter was palpable, and the upper pole of the right lobe
was extremely tender. Her serum free T4 and T3 levels were high, and the serum
TSH was suppressed to subnormal. She was positive for serum C-reactive protein.
Anti-thyroidal autoantibodies were all negative. Her thyrotoxicosis subsided
spontaneously within one week. Serum titers of antibodies to various viruses
were unchanged during the clinical course for two weeks, but she was positive
for HLA B35. Examination of a needle-biopsy specimen of the thyroid gland showed
extensive amyloid deposition and no evidence of subacute thyroiditis. We
considered her transient thyrotoxicosis to be associated with amyloid goiter.
The clinical course of this case was similar to the subacute thyroiditis-like
syndrome, first described by Ikenoue et al. When patients with primary or
secondary amyloidosis have symptoms and signs of subacute thyroiditis, but
develop an unusual course, amyloid goiter should be considered. To study the possible role of viral infection in the etiology of subacute
thyroiditis (SAT), we measured serum virus-specific antibodies to measles,
rubella, mumps, type I herpes, chicken pox, human parvovirus B19 and
cytomegalovirus (CMV) in 10 patients with SAT during the course of illness. In
spite of the presence of IgG to each virus in more than 70% of patients, no
significant changes in the IgG titers were observed except those to measles,
rubella, chicken pox or CMV in only 10% of patients, respectively. Then we
examined the presence of virus DNA in specimens of 9 patients with SAT obtained
by fine-needle aspiration biopsy (FNAB) of the thyroid. DNA was amplified to
detect that of Epstein-Barr virus and CMV by polymerase chain reaction. However,
none of them were detected in all the specimens. Whereas previous studies
suggest the involvement of viral infection in the pathogenesis of SAT, we failed
to demonstrate significant changes in serum antiviral antibody titers or to
detect viral DNA in the specimens obtained by FNAB of the thyroid. Thus further
studies are clearly required to establish the definite role of viral infection
in the pathogenesis of SAT. Author information:
(1)Department of Microbiology and Infectious Diseases, The Alfred Hospital,
Prahran, Victoria 3181, Australia. To clarify the cellular composition of subacute thyroiditis, histologic and
immunohistochemical studies were performed. Histologically, the lesion presented
a patchy distribution of non-caseous granulomas comprising colloid, small
lymphocytes, neutrophils, macrophages with or without epithelioid features, and
multinucleated giant cells of foreign body type. In addition, numerous
plasmacytoid monocytes were closely associated with the granulomas. The giant
cells were CD68+, thyroglobulin- and cytokeratin-. Usually, small lymphocytes in
the granulomas are CD3+, CD8+, CD45RO+ cytotoxic T-cells. In the
non-granulomatous lesion, the follicles were often infiltrated by CD8+
T-lymphocytes, plasmacytoid monocytes and histiocytes, resulting in disrupted
basement membrane and rupture of the follicles. Lymphoid follicles with or
without active germinal centers were not observed. Moreover, no residual
follicular dendritic cell networks were detected by CD23 and CAN.42
immunostains. In the interfollicular area, scattered plasma cells were observed
among infiltrating cells. Neither human herpes virus 8 nor EBER-positive cells
were detected in the six patients. The findings of our study suggest that
cellular immune response may play an important role in the pathogenesis of
subacute thyroiditis. Subacute thyroiditis is a rare disease in childhood and is considered postviral
in origin. Epstein-Barr virus (EBV) involvement has been suspected, but just
once demonstrated in an adult female. We report a case of subacute thyroiditis
during infectious mononucleosis in a 3-year-old girl. The diagnosis of
infectious mononucleosis was supported, in addition to the typical clinical
symptoms, by presence of EBV DNA both in plasma and leukocytes. Subacute
thyroiditis was diagnosed based on elevated thyroid hormone levels with
thyrotropin (TSH) suppression, high inflammation markers and almost absent
(99)TC uptake by thyroid. Moreover after 3 months from diagnosis thyroid
function went back to normal as well as the (99)TC uptake. OBJECTIVE: Subacute thyroiditis (SAT) is a transient inflammatory disease of the
thyroid. We evaluated the clinical characteristics based on laboratory and
imaging studies in patients with SAT before treatment.
PATIENTS: We reviewed the medical records of 852 patients (107 men and 745
women) with SAT who visited our thyroid clinic at Kuma Hospital from 1996
through 2004.
RESULTS: SAT developed most often in female patients aged 40 to 50 years, with
significant seasonal clusters during summer to early autumn. While the rates of
any virus infections and diseases did not differ from those in the general
population, recurrent episodes of SAT at intervals of 13.6+/-5.6 years accounted
for 1.6% of all cases. At the onset of SAT, 28.2% of patients had temperatures
greater than 38 degrees C and typical symptoms associated with thyrotoxicosis
developed in more than 60% of patients. Before treatment, most of the abnormal
laboratory findings associated with thyrotoxicosis, inflammation, and liver
dysfunction reached peak levels within 1 week after onset. Ultrasound
examination showed that half of the patients with unilateral thyroid pain
presented with bilateral hypoechogenic area in the thyroid and the rate of
bilateral hypoechogenic area tended to increase 2 months after onset.
CONCLUSION: Laboratory studies of thyroid dysfunction and inflammation related
to SAT presented peak levels within 1 week after onset. Author information:
(1)Department of Child Health, Sultan Qaboos University Hospital, Muscat, Oman. Viral infections are frequently cited as a major environmental factor involved
in subacute thyroiditis and autoimmune thyroid diseases This review examines the
data related to the role of viruses in the development of thyroiditis. Our
research has been focused on human data. We have reviewed virological data for
each type of thyroiditis at different levels of evidence; epidemiological data,
serological data or research on circulating viruses, direct evidence of thyroid
tissue infection. Interpretation of epidemiological and serological data must be
cautious as they don't prove that this pathogen is responsible for the disease.
However, direct evidence of the presence of viruses or their components in the
organ are available for retroviruses (HFV) and mumps in subacute thyroiditis,
for retroviruses (HTLV-1, HFV, HIV and SV40) in Graves's disease and for HTLV-1,
enterovirus, rubella, mumps virus, HSV, EBV and parvovirus in Hashimoto's
thyroiditis. However, it remains to determine whether they are responsible for
thyroid diseases or whether they are just innocent bystanders. Further studies
are needed to clarify the relationship between viruses and thyroid diseases, in
order to develop new strategies for prevention and/or treatment. de Quervain thyroiditis, also known as subacute thyroiditis, is a self-limited
inflammatory disease of the thyroid gland. It is extremely rare in children. The
hallmarks for diagnosis are painful thyroid enlargement, elevated inflammatory
markers, and decreased uptake of the thyroid gland on thyroid scintigraphy.
Viral infection has been proposed to be associated with de Quervain thyroiditis.
Coxsackie virus has been reported to be one of the viruses associated with the
disease. To our knowledge, childhood de Quervain thyroiditis associated with
hand-foot-mouth disease caused by coxsackie infection has never been reported.
We report a 2.7-year-old boy who presented with typical features of de Quervain
thyroiditis following hand-foot-mouth disease caused by coxsackie B4 infection.
He had a brief thyrotoxic phase initially, followed by transient hypothyroid
phase and euthyroidism thereafter. His thyroid scintigraphy showed a typical
faint uptake at the diagnosis, and an improvement of the thyroid scan and uptake
was shown 8 weeks later. He was treated with prednisolone and nearly complete
resolution was documented within 2 months. Careful evaluation of the patient led
to the correct diagnosis and appropriate management. We describe a case of quick recovery of myocardium damage in a 15-year-old
adolescent with subacute thyroiditis. After 1 week of admission, his
cardiovascular status began to show signs of improvement accompanied by the
recovery of electrocardiogram and indicators of myocardial damage. We speculate
that myocardium damage associated with subacute thyroiditis is a complication of
common virus, although we did not detect any abnormal virus antibody and
deoxyribonucleic acid in the patient's serum. Subacute (de Quervain's) thyroiditis is a rare but important cause of fever of
unknown origin. Most cases of subacute thyroiditis are caused by a variety of
viruses, for example, Coxsackie, cytomegalovirus, Epstein-Barr virus, and
adenovirus. Influenza immunization or infection may cause subacute thyroiditis.
We present the first reported case of a fever of unknown origin due to seasonal
influenza A in a 67-year-old woman. BACKGROUND: Oral glucocorticoids are administered in moderate and severe cases
of subacute thyroiditis (SAT), providing dramatic relief from pain and fever.
However, there have been no reports regarding the optimal dose of prednisolone
(PSL) for treatment of SAT. In this study, we used 15 mg/day of PSL as the
initial dosage and tapered it by 5 mg every 2 weeks. We assessed the
effectiveness of this treatment protocol.
METHODS: We examined 384 consecutive and untreated patients with SAT who visited
our thyroid clinic between February 2005 and December 2008. We excluded patients
who did not fit our protocol, and the final number of subjects was 219. When
patients complained of pain in their neck or C-reactive protein (CRP) was still
high, physicians were able to extend the tapering of the dose of PSL or increase
it at 2-week intervals. The endpoint of the study was the duration of the PSL
medication. We also compared the severity of thyrotoxicosis and rate of
hypothyroidism after SAT between the short medication group (patients who
recovered within 6 weeks) and long medication group (patients who recovered in
12 weeks or more).
RESULTS: The number of patients whose thyroiditis improved within 6 weeks and
did not recur was 113 (51.6%), and 61 (27.9%) improved within 7 to 8 weeks and
did not have a recurrence. The longest duration was 40 weeks. Seven patients
(3.2%) needed increases in the dosage of PSL. Thyroid hormone (free thyroxine
and free triiodothyronine) levels measured at the initial visit in the short
medication group were significantly higher than those in the long medication
group (p<0.05). Serum CRP, male-to-female ratio, body weight, and age showed no
differences between the two groups. There were no differences in the rate of
hypothyroidism after SAT between the two groups (p=0.0632).
CONCLUSIONS: The treatment protocol that we employed had 15 mg/day of PSL as the
initial dosage for the treatment of SAT, with tapering by 5 mg every 2 weeks,
and was effective and safe for Japanese patients. However, 20% of patients with
SAT needed longer than 8 weeks to recover from the inflammation. |
List core circadian clock genes. | The core circadian clock genes are CLOCK, BMAL1, Per, and Cry. | From single cell organisms to the most complex life forms, the 24-hour circadian
rhythm is important for numerous aspects of physiology and behavior such as
daily periodic fluctuations in body temperature and sleep-wake cycles.
Influenced by environmental cues - mainly by light input -, the central
pacemaker in the thalamic suprachiasmatic nuclei (SCN) controls and regulates
the internal clock mechanisms which are present in peripheral tissues. In order
to correlate modifications in the molecular mechanisms of circadian rhythm with
the pathophysiology of idiopathic hypersomnia, this study aimed to investigate
the dynamics of the expression of circadian clock genes in dermal fibroblasts of
idiopathic hypersomniacs (IH) in comparison to those of healthy controls (HC).
Ten clinically and polysomnographically proven IH patients were recruited from
the department of sleep medicine of the University Hospital of Muenster.
Clinical diagnosis was done by two consecutive polysomnographies (PSG) and
Multiple Sleep Latency Test (MSLT). Fourteen clinical healthy volunteers served
as control group. Dermal fibroblasts were obtained via punch biopsy and grown in
cell culture. The expression of circadian clock genes was investigated by
semiquantitative Reverse Transcriptase-PCR qRT-PCR analysis, confirming
periodical oscillation of expression of the core circadian clock genes BMAL1,
PER1/2 and CRY1/2. The amplitude of the rhythmically expressed BMAL1, PER1 and
PER2 was significantly dampened in dermal fibroblasts of IH compared to HC over
two circadian periods whereas the overall expression of only the key
transcriptional factor BMAL1 was significantly reduced in IH. Our study suggests
for the first time an aberrant dynamics in the circadian clock in IH. These
findings may serve to better understand some clinical features of the
pathophysiology in sleep - wake rhythms in IH. Author information:
(1)InsermCESP Center for Research in Epidemiology and Population Health, U1018,
Environmental Epidemiology of Cancer, Villejuif, FranceUniversité Paris-SudUMRS
1018, Villejuif, FranceBiostatistical UnitMRC, Cambridge, UKSchool of
Mathematics and PhysicsThe University of Queensland, St Lucia, Queensland,
AustraliaInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Epidemiology of Diabetes, Obesity and Chronic Kidney Disease Over
Lifecourse, Villejuif, FranceUniversité Paris DescartesINSERM UMR-S775
EPIGENETEC, Paris, FranceDépartement d'informatique médicaleCenter
Georges-François Leclerc, Dijon, FranceCenter Eugène MarquisRennes,
FranceInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Environmental Epidemiology of Cancer, Villejuif, FranceUniversité
Paris-SudUMRS 1018, Villejuif, FranceBiostatistical UnitMRC, Cambridge, UKSchool
of Mathematics and PhysicsThe University of Queensland, St Lucia, Queensland,
AustraliaInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Epidemiology of Diabetes, Obesity and Chronic Kidney Disease Over
Lifecourse, Villejuif, FranceUniversité Paris DescartesINSERM UMR-S775
EPIGENETEC, Paris, FranceDépartement d'informatique médicaleCenter
Georges-François Leclerc, Dijon, FranceCenter Eugène MarquisRennes, France.
(2)InsermCESP Center for Research in Epidemiology and Population Health, U1018,
Environmental Epidemiology of Cancer, Villejuif, FranceUniversité Paris-SudUMRS
1018, Villejuif, FranceBiostatistical UnitMRC, Cambridge, UKSchool of
Mathematics and PhysicsThe University of Queensland, St Lucia, Queensland,
AustraliaInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Epidemiology of Diabetes, Obesity and Chronic Kidney Disease Over
Lifecourse, Villejuif, FranceUniversité Paris DescartesINSERM UMR-S775
EPIGENETEC, Paris, FranceDépartement d'informatique médicaleCenter
Georges-François Leclerc, Dijon, FranceCenter Eugène MarquisRennes, France.
(3)InsermCESP Center for Research in Epidemiology and Population Health, U1018,
Environmental Epidemiology of Cancer, Villejuif, FranceUniversité Paris-SudUMRS
1018, Villejuif, FranceBiostatistical UnitMRC, Cambridge, UKSchool of
Mathematics and PhysicsThe University of Queensland, St Lucia, Queensland,
AustraliaInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Epidemiology of Diabetes, Obesity and Chronic Kidney Disease Over
Lifecourse, Villejuif, FranceUniversité Paris DescartesINSERM UMR-S775
EPIGENETEC, Paris, FranceDépartement d'informatique médicaleCenter
Georges-François Leclerc, Dijon, FranceCenter Eugène MarquisRennes,
FranceInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Environmental Epidemiology of Cancer, Villejuif, FranceUniversité
Paris-SudUMRS 1018, Villejuif, FranceBiostatistical UnitMRC, Cambridge, UKSchool
of Mathematics and PhysicsThe University of Queensland, St Lucia, Queensland,
AustraliaInsermCESP Center for Research in Epidemiology and Population Health,
U1018, Epidemiology of Diabetes, Obesity and Chronic Kidney Disease Over
Lifecourse, Villejuif, FranceUniversité Paris DescartesINSERM UMR-S775
EPIGENETEC, Paris, FranceDépartement d'informatique médicaleCenter
Georges-François Leclerc, Dijon, FranceCenter Eugène MarquisRennes, France
[email protected]. Circadian clock genes regulate 10-15% of the transcriptome, and might function
as tumor suppressor genes. Relatively little is known about the circadian clock
in tumors and its effect on surrounding healthy tissues. Therefore, we compared
the 24-hr expression levels of key circadian clock genes in liver and kidney of
healthy control mice with those of mice bearing C26 colorectal tumor metastases
in the liver. Metastases were induced by injection of C26 colorectal carcinoma
cells into the spleen. Subsequently, tumor, liver and kidney tissue was
collected around the clock to compare circadian rhythmicity. Expression levels
of five clock genes (Rev-Erbα, Per1, Per2, Bmal1 and Cry1) and three
clock-controlled genes (CCGs; Dbp, p21 and Wee1) were determined by qRT-PCR.
Liver and kidney tissue of healthy control mice showed normal 24-hr oscillations
of all clock genes and CCGs, consistent with normal circadian rhythmicity. In
colorectal liver metastases, however, 24-hr oscillations were completely absent
for all clock genes and CCGs except Cry1. Liver and kidney tissue of
tumor-bearing mice showed a shift in clock periodicity relative to control mice.
In the liver we observed a phase advance, whereas in the kidney the phase was
delayed. These data show that hepatic metastases of C26 colon carcinoma with a
disrupted circadian rhythm phase shift liver and kidney tissue clocks, which
strongly suggests a systemic effect on peripheral clocks. The possibility that
tumors may modify peripheral clocks to escape from ordinary circadian rhythms
and in this way contribute to fatigue and sleep disorders in cancer patients is
discussed. Author information:
(1)Department of Biochemistry and Biomedical Sciences, College of Medicine,
Seoul National University, 103 Daehak-ro, Seoul, 110-799, Jongno-gu, Korea.
[email protected].
(2)Department of Biochemistry, College of Medicine, Konyang University, Daejeon,
302-718, Korea. [email protected].
(3)Department of Biological Sciences, Seoul National University, Seoul, 151-742,
Korea. [email protected].
(4)Department of Neuroscience and Neurodegeneration Control Research Center,
Kyung Hee University, Seoul, 130-701, Korea. [email protected].
(5)Department of Biochemistry and Biomedical Sciences, College of Medicine,
Seoul National University, 103 Daehak-ro, Seoul, 110-799, Jongno-gu, Korea.
[email protected].
(6)Department of Biochemistry and Biomedical Sciences, College of Medicine,
Seoul National University, 103 Daehak-ro, Seoul, 110-799, Jongno-gu, Korea.
[email protected].
(7)Department of Neuroscience and Neurodegeneration Control Research Center,
Kyung Hee University, Seoul, 130-701, Korea. [email protected].
(8)Department of Brain Science, DGIST, Daegu, 711-873, Korea.
[email protected].
(9)Department of Biochemistry and Biomedical Sciences, College of Medicine,
Seoul National University, 103 Daehak-ro, Seoul, 110-799, Jongno-gu, Korea.
[email protected]. The cause of sudden sensorineural hearing loss (SSNHL) remains unclear and
therefore it is often considered as idiopathic. Sleep disturbance has been
linked to SSNHL and circadian rhythm disruption, but the link between circadian
rhythm disruption and SSNHL has never been investigated.In this study, we
surveyed the sleep quality of 38 patients with SSNHL using a simple insomnia
sleep questionnaire. The expression of circadian clock genes in peripheral blood
(PB) leukocytes from 38 patients with SSNHL and 71 healthy subjects was accessed
using real-time quantitative reverse transcriptase-polymerase chain reaction and
validated using immunocytochemical staining.We found that 61.8% of patients with
SSNHL suffered from insomnia before the insult of hearing loss. Besides,
significantly decreased expression of PER1, CRY1, CRY2, CLOCK, BMAL1, and CKlε
was found in PB leukocytes of patients with SSNHL when compared with healthy
subjects. SSNHL patients with vertigo had significantly lower expression of CRY1
and CKlε than patients without vertigo symptoms. Our results imply the
association of sleep disturbance and disrupted circadian rhythm in SSNHL. |
Which peripheral neuropathy has been associated with NDRG1 mutations? | Charcot-Marie-Tooth (CMT) 4D disease is a severe autosomal recessive demyelinating neuropathy with extensive axonal loss leading to early disability, caused by mutations in the N-myc downstream regulated gene 1 (NDRG1). | In a previous study, we have shown that N-myc downstream-regulated gene 1
(NDRG1), classified in databases as a tumor suppressor and heavy metal-response
protein, is mutated in hereditary motor and sensory neuropathy Lom (HMSNL), a
severe autosomal recessive form of Charcot-Marie-Tooth (CMT) disease. The
private founder mutation R148X, causing HMSNL in patients of Romani ethnicity,
has so far remained the only molecular defect linking NDRG1 to a specific
disease phenotype. Here we report the first study aiming to assess the overall
contribution of this gene to the pathogenesis of peripheral neuropathies, in
cases where the most common causes of CMT disease have been excluded. Sequence
analysis of NDRG1 in 104 CMT patients of diverse ethnicity identified one novel
disease-causing mutation, IVS8-1G>A (g.2290787G>A), which affects the
splice-acceptor site of IVS8 and results in the skipping of exon 9. The
phenotype of the IVS8-1G>A homozygote was very closely related to that of HMSNL
patients. In addition, we have detected homozygosity for the known R148X
mutation in two affected individuals. Mutations in NDRG1 thus accounted for
2.88% of our overall group of patients, and for 4.68% of cases with
demyelinating neuropathies. No other variants were identified in the coding
sequence, whereas 12 single nucleotide polymorphisms were observed in the
introns. Hum Mutat 22:129-135, 2003. Hereditary neuropathies are classified into several subtypes according to
clinical, electrophysiologic and pathologic findings. Recent genetic studies
have revealed their phenotypic and genetic diversities. In the primary
peripheral demyelinating neuropathies(CMT1), at least 9 genes have been
associated with the disorders; altered dosage of peripheral myelin protein
22(PMP22) or point mutation of PMP22, the gap junction protein 1(GJB1), the
myelin protein zero gene(MPZ), the early growth response gene 2(EGR2), the
myotubularin-related protein 2 gene(MTMR2), the N-myc downstream-regulated gene
1 (NDRG1), the L-periaxin gene(PRX), SRY-related HMG-BOX gene 10(SOX10) and the
ganglioside-induced differentiation-associated protein 1 gene(GDAP1). In the
primary peripheral axonal neuropathies(CMT2), at least 8 genes have been
associated with these disorders; the neurofilament light chain gene(NEFL), the
kinesin 1B gene(KIF1B), the gigaxonin gene(GAN1), Lamin A/C(LMNA) and
tyrosyl-DNA phosphodiesterase 1(TDP1). In addition, some mutations in GJB1, MPZ
and GDAP1 also present with clinical and electrophysiologic findings of CMT2.
Mutation of NEFL or KIF1B cause domitly inherited axonal neuropathies,
whereas mutation of GJB1 or MPZ can present as genocopies of domit axonal
neuropathies. In addition to the above diseases, we have reported a new type of
NMSNP(MIM # *604484) characterized by proximal domit neurogenic atrophy,
obvious sensory nerve involvement and the gene locus on 3q13. Here, we summarize
the genetic bases of hereditary neuropathies and attempt to highlight
significant genotype-phenotype correlations. NDRG1 is an intracellular protein that is induced under a number of stress and
pathological conditions, and it is thought to be associated with cell growth and
differentiation. Recently, human NDRG1 was identified as a gene responsible for
hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth
disease type 4D), which is characterized by early-onset peripheral neuropathy,
leading to severe disability in adulthood. In this study, we generated mice
lacking Ndrg1 to analyze its function and elucidate the pathogenesis of
Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the
sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5
weeks of age. However, myelination of Schwann cells in the sciatic nerve was
normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness,
especially in the hind limbs, but complicated motor skills were retained. In
wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells
rather than the myelin sheath. These results indicate that NDRG1 deficiency
leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for
maintece of the myelin sheaths in peripheral nerves. These mice will be used
for future analyses of the mechanisms of myelin maintece. Hereditary neuropathies are classified into several subtypes according to
clinical, electrophysiologic and pathologic findings. Recent genetic studies
have revealed their phenotypic and genetic diversities. In the primary
peripheral demyelinating neuropathies (CMT1), at least 15 genes have been
associated with the disorders; altered dosage or point mutation of PMP22, GJB1,
MPZ, EGR2, MTMR2, NDRG1, PRX, SOX10, GDAP1 and MTMR13/SBF2. In the primary
peripheral axonal neuropathies (CMT2), at least 10 genes have been associated
with these disorders; NEFL, KIF1B, MFN2, GAN1, LMNA, RAB7, GARS, TDP1, APTX, and
SETX. In addition, some mutations in GJB1, MPZ, GDAP1 and NEFL also present with
clinical and electrophysiologic findings of CMT2. Patients with TDP1, APTX or
SETX mutations share common clinical findings; autosomal recessive inheritance,
cerebellar ataxia, and axonal neuropathy. These genes are suspected to be
related to DNA/RNA repair and induce cell death especially in neuronal cells. In
addition to the above diseases, we have reported a new type of NMSNP (MIM# *
604484) characterized by proximal domit neurogenic atrophy, obvious sensory
nerve involvement and the gene locus on 3q12.3. Here, we summarize the genetic
bases of hereditary neuropathies and attempt to highlight significant
genotype-phenotype correlations with a special interest in nonsense-mediated
mRNA decay pathway. Inherited neuropathies are clinically and genetically heterogeneous. At least 28
genes and 12 loci have been associated with Charcot-Marie-Tooth disease (CMT)
and related inherited neuropathies. Most causes of inherited neuropathy have
been discovered by positional cloning technique and in the past two years, the
pace of CMT gene discovery has accelerated. Genetic studies have revealed the
following gene mutations as the causes of inherited neuropathies; PMP22, MPZ,
EGR2, SOX10, SIMPLE/LITAF, ARHGEF10 for CMT1 (autosomal domit demyelinating
form); GDAP1, MTMR2, SBF2/MTMR13, KIAA1985, NDRG1 PRX for CMT4 (autosomal
recessive demyelinating form), MFN2, KIF1B, RAB7, GARS, NEFL, HSPB1, HSPB8 for
CMT2 (autosomal domit axonal form); LMNA, GAN1, KCC3, TDP1, APTX, SETX for
AR-CMT2 (autosomal recessive axonal form); GIB1 for CMTX (X-linked CMT); DNM2
for CMT-DI (autosomal domit CMT with intermediate nerve conduction
velocities); and DHH for minifascicular neuropathy. These discovered CMT causing
genes/proteins include those which show unpredictable correlations with the
peripheral nervous system. However, these genes/proteins are definitely
important for the peripheral nerve, and their discovery should pave the way for
dramatic progress in the understanding of peripheral nerve biology. On the other
hand, genotype-phenotype correlations of these genes are also important in order
to understand the pathomechanisms of inherited neuropathy. Because, based on
mutation studies, a large number of genes associated with both the CMT1/4 and
CMT2 forms have been identified, it is usually difficult to predict the
causative gene based on clinical information from patients without specific
complications. To clarify the specific features and molecular mechanisms of five
diseases that we previously reported, we reviewed recent progress in HMSN-P
linked to chromosome 3, CMT4F caused by PRX, CMT4A caused by GDAP1, CMT4B2
caused by SBF2/MTMR13, and SCAN1 caused by TDP1. HMSN-P is characterized by late
onset, proximal domit severe muscle weakness, fasciculations, muscle cramp
and sensory involvement. HMSN-P is a primary neuronopathy. Mutations in periaxin
are associated with a broad spectrum of demyelinating neuropathies including
DSS, a sensory domit form and early onset slowly progressive CMT.
Pathologically, loss of myelinated fibers, demyelination, small onion bulb
formations, tomacula formation and myelin foldings were seen in sural nerves.
Absence of septate like junction in the paranodal loop suggests that periaxin
could be required for the adhesion complex. GDAP1 is a relatively common cause
of CMT4. Half of reported patients showed the demyelinating form, while the rest
showed the axonal form. The typical feature of CMT4A is paresis of the vocal
cords and diaphragm. CMT4B2 is characterized by autosomal recessive, juvenile
onset glaucoma and focally folded myelin in sural nerves. SBF2/MTMR13 mutations
cause CMT4B2. Early onset glaucoma was seen in patients with nonsense mutations.
SBF2/MTMR13 and MTMR2, which is the cause of CMT4B1, could be acting on the same
3-phosphoinositide signaling pathway. Clinical phenotypes of patients with TDP1,
APTX, or SETX mutations share common clinical findings, namely cerebellar ataxia
and axonal neuropathy. TDP1 and aprataxin both act on the single strand break
repair pathway, with TDP1 working specifically on topoisomerase I related SSBR.
Senataxin is a RNA helicase acting on RNA maturation and termination in yeast.
Since these three proteins share a common pathway, disruption in any of them
could induce a delay in the transcription process. The low rate of protein
supply could lead to deaths of large neuronal cells. The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities
to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans.
The pedigrees containing affected dogs suggest monogenic autosomal recessive
inheritance and all affected dogs trace back to a single male. Here, we studied
the neuropathology of this disease and identified a candidate causative
mutation. Peripheral nerve biopsies from affected dogs were examined using
semi-thin histology, nerve fibre teasing and electron microscopy. A severe
chronic progressive mixed polyneuropathy was observed. Seven affected and 17
related control dogs were genotyped on the 50k canine SNP chip. This allowed us
to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by
homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1
mutations have been shown to cause hereditary motor and sensory neuropathy-Lom
in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional
candidate gene and performed mutation analysis in affected and control
Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC)
was perfectly associated with the polyneuropathy phenotype of Greyhound show
dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several
amino acids before a stop codon is encountered. A reduced level of NDRG1
transcript could be detected by RT-PCR. Western blot analysis demonstrated an
absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We
thus have identified a candidate causative mutation for polyneuropathy in
Greyhounds and identified the first genetically characterized canine CMT model
which offers an opportunity to gain further insights into the pathobiology and
therapy of human NDRG1 associated CMT disease. Selection against this mutation
can now be used to eliminate polyneuropathy from Greyhound show dogs. CMT4D disease is a severe autosomal recessive demyelinating neuropathy with
extensive axonal loss leading to early disability, caused by mutations in the
N-myc downstream regulated gene 1 (NDRG1). NDRG1 is expressed at particularly
high levels in the Schwann cell (SC), but its physiological function(s) are
unknown. To help with their understanding, we characterise the phenotype of a
new mouse model, stretcher (str), with total Ndrg1 deficiency, in comparison
with the hypomorphic Ndrg1 knock-out (KO) mouse. While both models display
normal initial myelination and a transition to overt pathology between weeks 3
and 5, the markedly more severe str phenotype suggests that even low Ndrg1
expression results in significant phenotype rescue. Neither model replicates
fully the features of CMT4D: although axon damage is present, regenerative
capacity is unimpaired and the mice do not display the early severe axonal loss
typical of the human disease. The widespread large fibre demyelination coincides
precisely with the period of rapid growth of the animals and the dramatic
(160-500-fold) increase in myelin volume and length in large fibres. This is
followed by stabilisation after week 10, while small fibres remain unaffected.
Gene expression profiling of str peripheral nerve reveals non-specific secondary
changes at weeks 5 and 10 and preliminary data point to normal proteasomal
function. Our findings do not support the proposed roles of NDRG1 in growth
arrest, terminal differentiation, gene expression regulation and proteasomal
degradation. Impaired SC trafficking failing to meet the considerable demands of
nerve growth, emerges as the likely pathogenetic mechanism in NDRG1 deficiency. Autosomal recessive forms of Charcot-Marie-Tooth disease (CMT) account for less
than 10 % of all CMT cases, but are more frequent in the populations with a high
rate of consanguinity. Roma (Gypsies) are a transnational minority with an
estimated population of 10 to 14 million, in which a high degree of
consanguineous marriages is a generally known fact. Similar to the other
genetically isolated founder populations, the Roma harbour a number of unique or
rare autosomal recessive disorders, caused by "private" founder mutations. There
are three subtypes of autosomal recessive CMT with mutations private to the Roma
population: CMT4C, CMT4D and CMT4G. We report on the molecular examination of
four families of Roma origin in Slovakia with early-onset demyelinating
neuropathy and autosomal recessive inheritance. We detected mutation p.R148X
(g.631C>T) in the NDRG1 (NM_006096.3) gene in two families and mutation
g.9712G>C in the HK1 (NM_033498) gene in the other two families. These mutations
cause CMT4D and CMT4G, respectively. The success of molecular genetic analysis
in all families confirms that autosomal recessive forms of CMT caused by
mutations on the NDRG1 and HK1 genes are common causes of inherited neuropathies
among Slovak Roma. Providing genetic analysis of these genes for patients with
Roma origin as a common part of diagnostic procedure would contribute to a
better rate of diagnosed cases of demyelinating neuropathy in Slovakia and in
other countries with a Roma minority. |
Is miR-126 involved in heart failure? | Yes, miR-126 is associated with heart failure. | BACKGROUND: MicroRNAs (miRNAs) are endogenous small RNAs that are 21-25
nucleotides in length. Recently, plasma miRNAs have been reported to be
sensitive and specific biomarkers of various tissue injuries and pathological
conditions. The goal of this study was to assess plasma miRNA profiles and to
identify plasma miRNAs that are differentially expressed in patients with heart
failure.
METHODS AND RESULTS: A total of 33 patients with ischemic heart diseases and 17
asymptomatic controls were recruited. In 10 patients with heart failure, miRNAs
were assessed at both NYHA IV and III. miRNA array analyses were found to be not
appropriate for plasma miRNA profiling. The plasma concentrations of
well-characterized miRNAs (miR-126, 122 and 499) were assessed by a real-time
reverse transcription-polymerase chain reaction using an artificial small RNA as
an internal standard. Plasma concentrations of miR-126 were negatively
correlated with age and logBNP. In 10 patients with heart failure, plasma
concentrations of miR-126 were up-regulated with improvement of the NYHA class
from IV to III.
CONCLUSIONS: The plasma concentration of miR-126 was negatively correlated with
age and NYHA class, and could be a useful biomarker for heart failure. BACKGROUND: MicroRNA (miRNA) expression profiles in endothelial progenitor cells
(EPCs) contribute to EPC dysfunction in patients suffering from coronary artery
disease. However, it remains unclear whether miRNA expression in EPCs is
associated with the prognosis of chronic heart failure (CHF) secondary to
ischemic cardiomyopathy (ICM) or non-ischemic cardiomyopathy (NICM).
METHODS AND RESULTS: One hundred six patients with CHF (55 ICM and 51 NICM) and
30 healthy controls were followed until the end of 24 months or when the end
point was obtained (cardiovascular death). The miRNA expression profile was
analyzed by TaqMan Human MicroRNA Array Set v2.0 in 30 randomly assigned samples
(ICM=10, NICM=10, and healthy controls=10). During the 24-month follow-up, 26
patients died from cardiovascular disease. Sixteen miRNAs (miR-126, miR-508-5p,
miR-34a, miR-210, miR-490-3p, miR-513-5p, miR-517c, miR-518e, miR-589, miR-220c,
miR-200a*, miR-186*, miR-7i*, miR-200b*, miR-595, and miR-662) were found to be
differentially expressed between ICM and NICM patients. Survival analysis showed
that miR-126 and miR-508-5p levels in EPCs were independent prognostic factors
(P=0.003; HR (hazard ratio): 0.19; 95% CI (confidence intervals): 0.06-0.58,
P=0.002; HR: 2.292; 95% CI: 1.37-3.84) for the outcome of ICM or NICM patients
with CHF. Pathway enrichment analysis showed that the angiogenesis pathway was
the most likely pathway regulated by miR-126 and miR-508-5p.
CONCLUSIONS: The miRNAs miR-126 and miR-508-5p are associated with the outcome
of ICM and NICM patients with CHF. These two miRNAs could be useful in the
diagnosis of CHF patients, and might provide novel targets for prevention and
treatment of CHF. |
Does resveratrol reduce cardiac remodeling? | Resveratrol attenuates left ventricular remodeling.
Resveratrol is a beneficial pharmacological tool that augments autophagy to bring about reverse remodeling in the postinfarction heart.
Resveratrol administration improved cardiac environment by reducing inflammatory state and decreased unfavorable ventricular remodeling of the diabetic heart, leading to a marked recovery of ventricular function.
Resveratrol can constitute an adjuvant therapeutic option in prevention of dilated cardiomyopathy. | Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating,
differentiating, and secreting extracellular matrix proteins. Prolonged
activation of CFs leads to cardiac fibrosis and reduced myocardial contractile
function. Resveratrol (RES) exhibits a number of cardioprotective properties;
however, the possibility that this compound affects CF function has not been
considered. The current study tests whether RES directly influences the growth
and proliferation of CFs and differentiation to the hypersecretory myofibroblast
phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG
II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase
activation. This inhibition by RES reduced basal proliferation and blocked ANG
II-induced growth and proliferation of CFs in a concentration-dependent manner,
as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively.
RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with
0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK
activation over basal was similar to the phosphorylation induced by 100 nM ANG
II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM
ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II-
and transforming growth factor-beta-induced CF differentiation to the
myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin
expression and stress fiber organization in CFs. This study identifies RES as an
anti-fibrotic agent in the myocardium by limiting CF proliferation and
differentiation, two critical steps in the pathogenesis of cardiac fibrosis. We have previously shown that resveratrol possesses cardioprotective effect,
which may be attributed to its ability to (i) stimulate nitric oxide production
and (ii) free radical scavenging activity. Since resveratrol is one of the major
components of certain varieties of red grapes, these events may underlie the
cardioprotective effects thought to be obtained from moderate red wine
consumption. Here we report resveratrol enhanced myocardial angiogenesis both in
vivo and in vitro by induction of vascular endothelial growth factor (VEGF),
which was regulated by thioredoxin-1 (Trx-1) and heme oxygenase-1 (HO-1). Human
coronary arteriolar endothelial cells exposed to resveratrol or Trx-1 on
Matrigel demonstrated significantly accelerated tubular morphogenesis with
induction of HO-1 and VEGF expression. This angiogenic response was repressed by
tin-protoporphyrin IX (SnPP), an HO-1 inhibitor, along with downregulation of
VEGF expression. However Trx-1 expression was not affected by SnPP. Again, rat
neonatal cardiomyocytes treated with resveratrol significantly expressed Trx-1,
HO-1 as well as VEGF. Rats were orally administered with resveratrol (1 mg/kg
per day) for 14 days and then underwent permanent left anterior descending
coronary artery (LAD) occlusion to document similar pro-angiogenic effect. Our
results demonstrated that pretreatment with resveratrol markedly reduced infarct
size 24 h after myocardial infarction (MI) and increased capillary density in
the peri-infarct myocardium along with better left ventricular function 4 days
after MI compared with vehicle-treated control. Concomitantly,
resveratrol-treated myocardium after MI significantly induced Trx-1, HO-1 and
VEGF expression. This effect was blocked by SnPP. Our findings suggest that
resveratrol mediates cardioprotection and neovascularization through
Trx-1-HO-1-VEGF pathway in rat ischemic myocardium. This study was designed to examine the effects of the antioxidant resveratrol on
cardiac structure and function in pressure overload (PO)-induced cardiac
hypertrophy. Male Sprague-Dawley rats were subjected to sham operation and the
aortic banding procedure. A subgroup of sham control and aortic-banded rats were
treated with resveratrol for 2 wk after surgery. Echocardiographic analysis of
cardiac structure and function along with Western blot analysis of endothelial
nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and redox
factor-1 (ref-1) were performed in all groups after 4 wk of surgery. Banded rats
showed significantly increased left ventricle-to-body weight ratio.
Echocardiographic analysis showed that the interventricular septal wall
thickness and left ventricular posterior wall thickness at systole and diastole
were significantly increased in banded rats. Also, a significant increase in
isovolumic relaxation time was observed in banded rats. Measured eNOS, iNOS, and
ref-1 protein levels were significantly reduced in banded rats. Resveratrol
treatment prevented the above changes in cardiac structure, function, and
protein expression in banded rats. Aortic banding after 4 wk resulted in
concentric remodeling and impaired contractile function due to PO on the heart.
The 2-wk treatment with resveratrol was found to abolish PO-induced cardiac
hypertrophy. Resveratrol may therefore be beneficial against PO-induced cardiac
hypertrophy found in clinical settings of hypertension and aortic valve
stenosis. Resveratrol (RES; trans-3,5,4'-trihydroxystilbene) has been shown to improve
health and slow the progression of disease in various models. Several
cardioprotective mechanisms have been identified including antioxidant,
anti-inflammatory, and antifibrotic actions. Each of these actions is thought to
have the ability to attenuate the pathophysiology underlying the deleterious
cardiac structural remodeling that results from acute myocardial infarction
(MI). Therefore, we evaluated the effect of resveratrol treatment on the
progression of cardiac remodeling after MI. Four groups of rats (sham, n = 6;
sham + RES, n = 21; MI, n = 26; MI + RES, n = 24) were treated for 13 weeks,
starting 7 days before ligation of the left anterior descending coronary artery.
Serial transthoracic echocardiography revealed that resveratrol had no effect on
MI-induced left-ventricular and left-atrial dilatation or reduction in
left-ventricular fractional shortening. Consistent with these findings,
resveratrol did not improve the deterioration of hemodynamic function or reduce
infarct size at 12 weeks post-MI. Resveratrol-treated animals did, however, show
preserved cardiac contractile reserve in response to dobutamine administration.
Radioligand binding revealed that MI reduced beta-adrenergic receptor density.
Resveratrol administration increased beta-adrenoceptor density, so that
resveratrol-treated MI rats had beta-adrenoceptor densities similar to normal
rats. Real-time reverse transcription-polymerase chain reaction revealed that
MI-induced changes in sarcoplasmic reticulum Ca2+-ATPase 2 and transforming
growth factor beta-1 expression were unaltered by resveratrol, whereas
MI-induced increases in atrial natriuretic factor (ANF) and connective tissue
growth factor (CTGF) expression were attenuated. Resveratrol treatment does not
improve cardiac remodeling and global hemodynamic function post-MI but does
preserve contractile reserve and attenuate ANF and CTGF up-regulation. We investigated the expression of the proinflammatory cytokine interleukin
(IL)-17 in cardiac fibroblasts and its induction by high glucose (HG). Our
results show that primary mouse cardiac fibroblasts (mCFs) secrete low basal
levels of IL-17 and that HG (25 mM D-glucose) as opposed to low glucose (5 mM
D-glucose + 20 mM mannitol) significantly enhances its secretion. HG induces
IL-17 mRNA expression by both transcriptional and posttranscriptional
mechanisms. HG induces phosphoinositide 3- kinase [PI3K; inhibited by adenoviral
(Ad).domit negative (dn)PI3Kp85], Akt (inhibited by Ad.dnAkt1), and ERK
(inhibited by PD-98059) activation and induces IL-17 expression via
PI3K-->Akt-->ERK-dependent signaling. Moreover, mCFs express both IL-17
receptors A and C, and although IL-17RA is upregulated, HG fails to modulate
IL-17RC expression. Furthermore, IL-17 stimulates net collagen production by
mCFs. Pretreatment with the phytoalexin resveratrol blocks HG-induced PI3K-,
Akt-, and ERK-dependent IL-17 expression. These results demonstrate that 1)
cardiac fibroblasts express IL-17 and its receptors; 2) HG upregulates IL-17 and
IL-17RA, suggesting a positive amplification loop in IL-17 signaling in
hyperglycemia; 3) IL-17 enhances net collagen production; and 4) resveratrol can
inhibit these HG-induced changes. Thus, in hyperglycemic conditions, IL-17 may
potentiate myocardial inflammation, injury, and remodeling through autocrine and
paracrine mechanisms, and resveratrol has therapeutic potential in ameliorating
this effect. It remains presently unknown whether vascular reactivity is impaired and whether
maladaptive cardiac remodeling occurs before the onset of overt obesity and in
the absence of hyperlipidemia. Normal female rats were fed a high-fat diet for 8
weeks and were associated with a modest nonsignificant increase of body weight
(standard diet, 300 +/- 10, versus high-fat diet, 329 +/- 14 g) and a normal
plasma lipid profile. In rats fed a high-fat diet, systolic (171 +/- 7 mm Hg)
and diastolic blood pressures (109 +/- 3) were increased compared to a standard
diet (systolic blood pressure, 134 +/- 8; diastolic blood pressure, 96 +/- 5 mm
Hg), and acetylcholine-dependent relaxation of isolated aortic rings (high-fat
diet, 22 +/- 5%, versus standard diet, 53 +/- 8%) was significantly reduced.
Furthermore, perivascular fibrosis was detected in the heart of rats fed a
high-fat diet. The exogenous addition of resveratrol
(trans-3,5,4'-trihydroxystilbene) (0.1 microM) to aortic rings isolated from
rats fed a high-fat diet restored acetylcholine-mediated relaxation (47 +/- 9%).
The administration of resveratrol (20 mg/kg/day for 8 weeks) to rats fed a
high-fat diet prevented the increase in blood pressure and preserved
acetylcholine-dependent relaxation of isolated aortic rings. However,
resveratrol therapy failed to attenuate the perivascular fibrotic response.
These data have demonstrated that a high-fat diet fed to normal female rats can
elicit a hypertensive response and induce perivascular fibrosis before the
development of overt obesity and in the absence of hyperlipidemia. Resveratrol
therapy can prevent the hypertensive response in female rats fed a high-fat diet
but is without effect on the progression of perivascular fibrosis. Whereas studies involving animal models of cardiovascular disease demonstrated
that resveratrol is able to inhibit hypertrophic growth, the mechanisms involved
have not been elucidated. Because studies in cells other than cardiomyocytes
revealed that AMP-activated protein kinase (AMPK) and Akt are affected by
resveratrol, we hypothesized that resveratrol prevents cardiac myocyte
hypertrophy via these two kinase systems. Herein, we demonstrate that
resveratrol reduces phenylephrine-induced protein synthesis and cell growth in
rat cardiac myocytes via alterations of intracellular pathways involved in
controlling protein synthesis (p70S6 kinase and eukaryotic elongation factor-2).
Additionally, we demonstrate that resveratrol negatively regulates the
calcineurin-nuclear factor of activated T cells pathway thus modifying a
critical component of the transcriptional mechanism involved in pathological
cardiac hypertrophy. Our data also indicate that these effects of resveratrol
are mediated via AMPK activation and Akt inhibition, and in the case of AMPK, is
dependent on the presence of the AMPK kinase, LKB1. Taken together, our data
suggest that resveratrol exerts anti-hypertrophic effects by activating AMPK via
LKB1 and inhibiting Akt, thus suppressing protein synthesis and gene
transcription. The purpose of this study was to investigate the effect of resveratrol, a
polyphenol present in grapes and red wine, on ventricular remodeling after
myocardial infarction (MI) in rats. After permanent ligation of the left
anterior descending artery, surviving rats were randomly allocated to three
groups and treated with 1 mg/kg/day resveratrol (R-1 group), 0.1 mg/kg/day
resveratrol (R-0.1 group), or vehicles (control group) administered by
intraperitoneal injection once daily for four weeks. We examined the effects of
resveratrol by echocardiography, hemodynamic studies, histologic examinations,
and real-time quantitative polymerase chain reaction. The R-1 group had
significantly increased fractional shortening of the left ventricle, ameliorated
left ventricular dilatation, reduced left ventricular end-diastolic pressure,
and reduced infarct size. In contrast, the R-0.1 group experienced no beneficial
effects on myocardial infarction. The R-1 group also had significantly
attenuated expression of myocardial atrial natriuretic peptide and transforming
growth factor-beta1 mRNAs. This study indicates that resveratrol is a potent
cardioprotective agent in MI rats. Its cardioprotective effects may be due to a
reduction of atrial natriuretic peptide and transforming growth factor-beta1,
which are known to protect the heart from detrimental remodeling. Cardiac fibroblasts are the most prevalent cell type in the heart and play a key
role in regulating normal myocardial function and in the adverse myocardial
remodeling that occurs with hypertension, myocardial infarction and heart
failure. Many of the functional effects of cardiac fibroblasts are mediated
through differentiation to a myofibroblast phenotype that expresses contractile
proteins and exhibits increased migratory, proliferative and secretory
properties. Cardiac myofibroblasts respond to proinflammatory cytokines (e.g.
TNFalpha, IL-1, IL-6, TGF-beta), vasoactive peptides (e.g. angiotensin II,
endothelin-1, natriuretic peptides) and hormones (e.g. noradrenaline), the
levels of which are increased in the remodeling heart. Their function is also
modulated by mechanical stretch and changes in oxygen availability (e.g.
ischaemia-reperfusion). Myofibroblast responses to such stimuli include changes
in cell proliferation, cell migration, extracellular matrix metabolism and
secretion of various bioactive molecules including cytokines, vasoactive
peptides and growth factors. Several classes of commonly prescribed therapeutic
agents for cardiovascular disease also exert pleiotropic effects on cardiac
fibroblasts that may explain some of their beneficial outcomes on the remodeling
heart. These include drugs for reducing hypertension (ACE inhibitors,
angiotensin receptor blockers, beta-blockers), cholesterol levels (statins,
fibrates) and insulin resistance (thiazolidinediones). In this review, we
provide insight into the properties of cardiac fibroblasts that underscores
their importance in the remodeling heart, including their origin,
electrophysiological properties, role in matrix metabolism, functional responses
to environmental stimuli and ability to secrete bioactive molecules. We also
review the evidence suggesting that certain cardiovascular drugs can reduce
myocardial remodeling specifically via modulatory effects on cardiac
fibroblasts. BACKGROUND: Cardiac hypertrophy is a compensatory enlargement of the heart in
response to stress such as hypertension. It is beneficial in reducing stress
placed on the heart. However, when the stress is of a chronic nature, it becomes
pathological and leads to cardiac dysfunction and heart failure. Current
treatments for hypertension and heart failure have proven beneficial but are not
highly specific and associated with side effects. Accordingly, there is an
important need for alternative strategies to provide safe and effective
treatment.
METHODS: Ten-week-old male spontaneously hypertensive rats (SHRs) and
Wistar-Kyoto (WKY) rats were treated with resveratrol (2.5 mg/kg/day) for a
period of 10 weeks. Systolic blood pressure, and cardiac structure and function
were measured in all groups at different time points of resveratrol treatment.
Oxidative stress was also determined in all groups after 10 weeks of resveratrol
treatment.
RESULTS: SHRs were characterized with high blood pressure and concentric
hypertrophy from 15 weeks of age. Cardiac functional abnormalities were also
evident in SHR from 15 weeks onwards. Resveratrol treatment significantly
prevented the development of concentric hypertrophy, and systolic and diastolic
dysfunction in SHR without lowering blood pressure. Resveratrol also
significantly reduced the oxidative stress levels of cardiac tissue in SHR.
CONCLUSIONS: Resveratrol treatment was beneficial in preventing the development
of concentric hypertrophy and cardiac dysfunction in SHR. The cardioprotective
effect of resveratrol in SHR may be partially mediated by a reduction in
oxidative stress. Thus, resveratrol may have potential in preventing cardiac
impairment in patients with essential hypertension. AIMS: On the basis of our previous reports that cardioprotection induced by
ischaemic preconditioning induces autophagy and that resveratrol, a polyphenolic
antioxidant present in grapes and red wine induces preconditioning-like effects,
we sought to determine if resveratrol could induce autophagy.
METHODS AND RESULTS: Resveratrol at lower doses (0.1 and 1 microM in H9c2
cardiac myoblast cells and 2.5 mg/kg/day in rats) induced cardiac autophagy
shown by enhanced formation of autophagosomes and its component LC3-II after
hypoxia-reoxygenation or ischaemia-reperfusion. The autophagy was attenuated
with the higher dose of resveratrol. The induction of autophagy was correlated
with enhanced cell survival and decreased apoptosis. Treatment with rapamycin
(100 nM), a known inducer of autophagy, did not further increase autophagy
compared with resveratrol alone. Autophagic inhibitors, wortmannin (2 microM)
and 3-methyladenine (10 mM), significantly attenuated the resveratrol-induced
autophagy and induced cell death. The activation of mammalian target of
rapamycin (mTOR) was differentially regulated by low-dose resveratrol, i.e. the
phosphorylation of mTOR at serine 2448 was inhibited, whereas the
phosphorylation of mTOR at serine 2481 was increased, which was attenuated with
a higher dose of resveratrol. Although resveratrol attenuated the activation of
mTOR complex 1, low-dose resveratrol significantly induced the expression of
Rictor, a component of mTOR complex 2, and activated its downstream survival
kinase Akt (Ser 473). Resveratrol-induced Rictor was found to bind with mTOR.
Furthermore, treatment with Rictor siRNA attenuated the resveratrol-induced
autophagy.
CONCLUSION: Our results indicate that at lower dose, resveratrol-mediated cell
survival is, in part, mediated through the induction of autophagy involving the
mTOR-Rictor survival pathway. Reduced sarcoplasmic calcium ATPase (SERCA2a) expression has been shown to play
a significant role in the cardiac dysfunction in diabetic cardiomyopathy. The
mechanism of SERCA2a repression is, however, not known. This study was designed
to examine the effect of resveratrol (RSV), a potent activator of SIRT1, on
cardiac function and SERCA2a expression in chronic type 1 diabetes. Adult male
mice were injected with streptozotocin (STZ) and fed with either a regular diet
or a diet enriched with RSV. STZ administration produced progressive decline in
cardiac function, associated with markedly reduced SERCA2a and SIRT1 protein
levels and increased collagen deposition; RSV treatment to these mice had a
tremendous beneficial effect both in terms of improving SERCA2a expression and
on cardiac function. In cultured cardiomyocytes, RSV restored SERCA2 promoter
activity, which was otherwise highly repressed in high-glucose media. Protective
effects of RSV were found to be dependent on its ability to activate Silent
information regulator (SIRT) 1. In cardiomyocytes, overexpression of SIRT1 was
found sufficient to activate SERCA2 promoter in a dose-dependent manner. In
contrast, pretreatment of cardiomyocytes with SIRT1 antagonist, splitomycin,
blocked these beneficial effects of RSV. In addition, SIRT1 knockout (+/-) mice
were also found to be more sensitive to STZ-induced decline in SERCA2a mRNA. The
data demonstrate that, in chronic diabetes, 1) the enzymatic activity of cardiac
SIRT1 is reduced, which contributes to reduced expression of SERCA2a and 2)
through activation of SIRT1, RSV enhances expression of SERCA2a and improves
cardiac function. Red wine contains many compounds that may have therapeutic use, including
resveratrol (3,4',5-trihydroxytrans-stilbene). Since resveratrol could be
administered both in the diet and as a therapeutic agent, defining appropriate
concentrations requires understanding of the pharmacokinetics. Resveratrol
absorption is rapid but plasma concentrations are low as it is rapidly and
efficiently converted into relatively hydrophilic phase-2 conjugates, and
metabolites, which are then rapidly excreted via the urine and bile. Resveratrol
is an effective antioxidant in vivo by increasing NO synthesis and also
maintaining the reduced intracellular redox state via the thioredoxin system.
Further, activation of sirtuins (one class of lysine deacetylases) may mediate
the cardiovascular responses shown by resveratrol. Studies on animal models of
human disease suggest that resveratrol has the potential to decrease
cardiovascular symptoms in patients with myocardial infarction, arrhythmias,
hypertension, cardiomyopathies, fibrosis, atherosclerosis, thrombosis and
diabetes, but, as yet, human clinical trials are rare. Cardioprotection by
resveratrol in rodent models may rely on mechanisms producing pharmacological
preconditioning in the heart including reducing reactive oxygen species,
improving vasorelaxation and angiogenesis, preventing inflammation and
apoptosis, delaying atherosclerosis as well as decreasing cardiovascular
remodelling. Interventional studies in humans need to be completed before
resveratrol can be considered as a standard therapeutic agent. Therefore, future
studies should focus on obtaining the level of evidence required to determine
whether resveratrol can be added to the list of evidence-based therapies for
cardiovascular diseases that includes renin-angiotensin system inhibitors,
beta-adrenoceptor antagonists and calcium entry blockers. To study the efficiency of maintaining the reduced tissue environment via
pre-treatment with natural antioxidant resveratrol in stem cell therapy, we
pre-treated male Sprague-Dawley rats with resveratrol (2.5 mg/kg/day gavaged for
2 weeks). After occlusion of the left anterior descending coronary artery (LAD),
adult cardiac stem cells stably expressing EGFP were injected into the border
zone of the myocardium. One week after the LAD occlusion, the cardiac reduced
environment was confirmed in resveratrol-treated rat hearts by the enhanced
expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector
factor-1 (Ref-1). In concert, cardiac functional parameters (left ventricular
ejection fraction and fractional shortening) were significantly improved. The
improvement of cardiac function was accompanied by the enhanced stem cell
survival and proliferation as demonstrated by the expression of cell
proliferation marker Ki67 and differentiation of stem cells towards the
regeneration of the myocardium as demonstrated by the enhanced expression of
EGFP 28 days after LAD occlusion in the resveratrol-treated hearts. Our results
demonstrate that resveratrol maintained a reduced tissue environment by
overexpressing Nrf2 and Ref-1 in rats resulting in an enhancement of the cardiac
regeneration of the adult cardiac stem cells as demonstrated by increased cell
survival and differentiation leading to cardiac function. Oxidative stress and inflammatory response induced by myocardial infarction play
important roles in the development of sympathetic neural remodeling. The present
study was designed to investigate whether resveratrol can improve sympathetic
neural remodeling and hence cause less arrhythmias via its anti-oxidant and
anti-inflammatory effects. Male Sprague Dawley rats were randomly assigned to
either vehicle or resveratrol (1 mg/kg) treatment for 4 weeks post myocardial
infarction. Another group of sham operated rats served as controls. Cardiac
electrophysiology examination was performed to evaluate the severity of
ventricular arrhythmias. Sympathetic neural remodeling characterized by
heterogeneous nerve sprouting and sympathetic hyperinnervation was assessed by
immunohistochemistry study. Western blotting and ELISA were used to evaluate
inflammatory responses and oxidative stress was also quantified. Resveratrol
treatment resulted in less episodes of inducible ventricular arrhythmias which
was closely associated with attenuated sympathetic neural remodeling (P<0.001,
respectively). Decreased nerve growth factor (NGF) expression was also observed
in resveratrol treated rats in the peri-infarct area at 4 weeks after myocardial
infarction (P<0.001). Interestingly, beneficial effects of resveratrol were also
associated with less inflammatory responses and oxidative stress. Our data
indicated that resveratrol can suppress sympathetic neural remodeling process
after myocardial infarction via attenuated inflammatory responses and oxidative
stress, which in turn leads to less inducibility of ventricular arrhythmias. The phytoalexin resveratrol (3,4',5-trihydroxy-trans-stilbene) may attenuate
cardiovascular disease in man. This study has determined whether treatment with
resveratrol (1 mg/kg/day orally) prevented cardiac fibrosis and the decreased
cardiovascular function in the DOCA-salt hypertensive rat as a model of human
hypertension. Uninephrectomised rats (UNX) administered DOCA (25mg every 4th day
sc) and 1% NaCl in drinking water for 28 days developed cardiac and vascular
remodelling. In these DOCA-salt rats, resveratrol decreased inflammatory cell
infiltration, decreased cardiac fibrosis (left ventricular interstitial and
perivascular collagen content) and improved cardiac and vascular function.
Resveratrol attenuated other features of cardiovascular remodelling such as
increases in systolic blood pressure, left ventricular wet weight, left
ventricular wall thickness, diastolic stiffness constant, as well as decreased
cardiac contractility and prolonged action potential duration characteristic of
DOCA-salt rats. In summary, resveratrol, at a nutritionally relevant dose,
prevents or attenuates the adverse changes in the cardiovascular system. We
propose that the anti-inflammatory and anti-fibrotic effects of resveratrol are
responsible, at least in part, for its amelioration in cardiovascular
remodelling in DOCA-salt rats. These actions of resveratrol could play an
important role in the protective effects on the human cardiovascular system
reported for this constituent of red wine. OBJECTIVES: The putative protective effects of resveratrol against oxidative
injury in the heart, kidney and brain tissues of rats induced with the
two-kidney, one-clip (2K1C) hypertension model were investigated.
METHODS: Wistar albino rats were divided into sham-operated (n = 8) or 2K1C
groups, in which rats received either resveratrol (10 mg/kg per day, i.p., n =
8), or saline (n = 8) starting at Week 3 after the surgery and continuing for
the following 6 weeks. Indirect blood pressure recordings and echocardiographic
images were made to evaluate cardiac function. At the end of Week 9 the animals
were decapitated and plasma, heart, kidney and brain were taken for biochemical
assays, while aortic rings were prepared for vascular reactivity studies.
KEY FINDINGS: 2K1C hypertension resulted in increased blood pressure, aortic
hypercontractility and reduced left ventricular function, leading to increased
lipid peroxidation and myeloperoxidase activity, concomitant with significant
reductions in tissue glutathione, superoxide dismutase, Na+/K+-ATPase and
catalase activities in the cardiac, renal and brain tissues, indicating the
presence of oxidative tissue damage in peripheral target organs. Elevated plasma
levels of lactate dehydrogenase, creatine kinase, as well as reduced plasma
levels of antioxidant capacity and nitric oxide further verified the severity of
oxidative injury. A 6-week treatment with resveratrol reversed all the measured
parameters, ameliorated hypertension-induced oxidative injury in the target
organs and improved cardiovascular function.
CONCLUSIONS: Resveratrol improved cardiovascular function through the
augmentation of endogenous antioxidants and the inhibition of lipid peroxidation
by maintaining a balance in oxidant/antioxidant status, which also ameliorated
hypertension-induced oxidative injury in the cardiac, renal and cerebral
tissues. Resveratrol, a constituent of red wine, and γ-tocotrienol, a constituent of palm
oil are important for cardioprotection. Although microRNAs are known regulators
for genes involved in cardiac remodelling, the regulatory pathway involving
microRNA has not been studied so far. We explored the cardioprotection by
resveratrol, longevinex and γ-tocotrienol in ischaemia/reperfusion(I/R) model of
rat and determined miRNA profile from isolated RNA. Systemic analyses of miRNA
array and theirs targets were determined using a number of computational
approaches. Resveratrol and γ-tocotrienol, either alone or in combination,
modulated the expression pattern of miRNAs close to the control level based on
PCA analyses. Differential expression was observed in over 75 miRNAs, some of
them, such as miR-21 and miR-20b (anti-angiogenic) were previously implicated in
cardiac remodelling. The target genes for the highest differentially expressed
miRNA include genes of various molecular functions such as TGFβ1-Smad3
signalling pathway, inflammation and their transcription factors, which may play
key role in reducing I/R injury. Administration of antagomiR-20 attenuated I/R
induced vascular endothelial growth factor and HIF1α level. All the
interventions treated for 3 weeks lead to significant cardioprotection against
ischaemia/reperfusion injury. A unique signature of miRNA profile is observed in
control heart pretreated with resveratrol or γ-tocotrienol. We have determined
specific group of miRNA in heart that have altered during IR injuries. Most of
those altered microRNA expressions modulated close to their basal level in
resveratrol or longevinex treated I/R rat. Interestingly, resveratrol and
γ-tocotrienol resulted in synergestic action. Our prior study had shown that resveratrol was a potent cardioprotective agent
in rats with myocardial infarction (MI). In this study, we further evaluated the
mechanism of cardioprotection of resveratrol by proteomic analysis. After
permanent ligation of the left anterior descending artery under isoflurane
anesthesia, surviving rats were randomly allocated to three groups and treated
with resveratrol at 1 mg/kg/day (MI/R group), or vehicles (sham group and MI
group) once daily for four weeks. In proteomic analysis, the MI group showed
decreased expression of adenylate kinase 1 (AK1) and mitochondrial
NADP⁺-dependent isocitrate dehydrogenase (IDPm) after MI compared with the sham
group. These variations were reversed by resveratrol in the MI/R group.
Validation with Western blot and immunohistochemical analyses showed similar
trends in protein expression profiling. Our studies suggest that the beneficial
effects of resveratrol on ventricular modeling may be due to increased
expression of AK1 and IDPm, which have been known to increase myocardial
energetic efficiency and reduce reactive oxygen species-mediated damage,
respectively. Exercise training (ET) improves endurance capacity by increasing both skeletal
muscle mitochondrial number and function, as well as contributing to favourable
cardiac remodelling.Interestingly, some of the benefits of regular exercise can
also be mimicked by the naturally occurring polyphenol, resveratrol (RESV).
However, it is not known whether RESV enhances physiological adaptations to ET.
To investigate this, male Wistar rats were randomly assigned to a control chow
diet or a chow diet that contained RESV (4 g kg⁻¹ of diet) and subsequently
subjected to a programme of progressive treadmill running for 12 weeks.
ET-induced improvements in exercise performance were enhanced by 21% (P <0.001)
by the addition of RESV to the diet. In soleus muscle, ET+RESV increased both
the twitch (1.8-fold; P <0.05) and tetanic(1.2-fold; P <0.05) forces generated
during isometric contraction, compared to ET alone. In vivo echocardiography
demonstrated that ET+RESV also increased the resting left ventricular ejection
fraction by 10% (P <0.05), and reduced left ventricular wall stress compared to
ET alone.These functional changes were accompanied by increased cardiac fatty
acid oxidation (1.2-fold;P <0.05) and favourable changes in cardiac gene
expression and signal transduction pathways that optimized the utilization of
fatty acids in ET+RESV compared to ET alone. Overall, our findings provide
evidence that the capacity for fatty acid oxidation is augmented by the addition
of RESV to the diet during ET, and that this may contribute to the improved
physical performance of rats following ET. We investigated the effect of resveratrol, a popular natural polyphenolic
compound with antioxidant and proautophagic actions, on postinfarction heart
failure. Myocardial infarction was induced in mice by left coronary artery
ligation. Four weeks postinfarction, when heart failure was established, the
surviving mice were started on 2-week treatments with one of the following:
vehicle, low- or high-dose resveratrol (5 or 50 mg/kg/day, respectively),
chloroquine (an autophagy inhibitor), or high-dose resveratrol plus chloroquine.
High-dose resveratrol partially reversed left ventricular dilation (reverse
remodeling) and significantly improved cardiac function. Autophagy was augmented
in those hearts, as indicated by up-regulation of myocardial
microtubule-associated protein-1 light chain 3-II, ATP content, and autophagic
vacuoles. The activities of AMP-activated protein kinase and silent information
regulator-1 were enhanced in hearts treated with resveratrol, whereas Akt
activity and manganese superoxide dismutase expression were unchanged, and the
activities of mammalian target of rapamycin and p70 S6 kinase were suppressed.
Chloroquine elicited opposite results, including exacerbation of cardiac
remodeling associated with a reduction in autophagic activity. When resveratrol
and chloroquine were administered together, the effects offset one another. In
vitro, compound C (AMP-activated protein kinase inhibitor) suppressed
resveratrol-induced autophagy in cardiomyocytes, but did not affect the events
evoked by chloroquine. In conclusion, resveratrol is a beneficial
pharmacological tool that augments autophagy to bring about reverse remodeling
in the postinfarction heart. Cardiac hypertrophy and associated myocardial remodeling is one of the main
complications of hypertension resulting in the development of heart failure. It
is of great significance to explore novel treatments to reverse cardiac
hypertrophy in hypertensives with or without affecting blood pressure. In the
present study, we investigated whether low-dose resveratrol alone or in a
combination with a blood pressure-lowering agent can reverse
hypertension-induced cardiovascular dysfunction. Twenty-week-old male
spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats were treated with
resveratrol (2.5 mg kg⁻¹ per day) and/or hydralazine (25 mg kg⁻¹ per day) for 8
weeks. Blood pressure, cardiac structure and function, and electrocardiogram
measurements were examined. Pressure myography of resistance arteries,
histological examinations of heart tissues, oxidative stress and inflammatory
measurements were also preformed to assess the efficacy of the treatment.
Although resveratrol treatment alone was ineffective in reducing systolic blood
pressure, diastolic blood pressure, diastolic dysfunction and vascular
remodeling, it significantly prevented the systolic impairment and reduced
myocardial fibrosis, and reduced oxidative stress and inflammation in
hypertensive rats. Furthermore, a combination of resveratrol with hydralazine
treatment significantly reduced blood pressure, improved systolic and diastolic
function, decreased fibrosis and improved vascular geometry. In summary,
low-dose resveratrol itself was unable to reduce systolic blood pressure,
diastolic blood pressure, diastolic dysfunction and vascular remodeling.
However, resveratrol alone alleviated cardiac fibrosis and some of the
functional abnormalities in SHRs. And a combination of resveratrol with
hydralazine was more effective than resveratrol or hydralazine alone in
improving overall cardiovascular parameters. The objective of this study was to investigate left cardiac damage and the
cardioprotective effects of resveratrol in old rats with COPD. Rats 22 months
old were divided into three groups: control (CTL), smoking and
lipopolysaccharides (SM/LPS), and SM/LPS plus resveratrol (SM/LPS-Res). Cardiac
function, pathology, oxidative stress, and apoptosis index were measured.
Expression of myocardial SIRT1 was studied by real-time quantitative polymerase
chain reaction (PCR) and Western blot detection. The heart weight-body weight
ratio (LVW/BW) increased in the SM/LPS group compared with the CTL group. Both
the LVW/BW and the area of fibrosis in the SM/LPS-Res group decreased compared
with those in the SM/LPS group. 8-OHdG expression increased in cardiac tissue of
rats in the SM/LPS group, which could be inhibited by resveratrol. Resveratrol
significantly increased the activity of superoxide dismutase (SOD) and reduced
the cardiac malonyldialdehyde (MDA) level in the SM/LPS-Res group. There was a
significant decrease in the extent of cardiomyocyte apoptosis in the SM/LPS-Res
group compared with the SM/LPS group. SIRT1 mRNA increased in the SM/LPS-Res
group compared with the SM/LPS group. In conclusion, resveratrol attenuated
cardiac oxidative damage and left ventricular remodeling and enhanced the
decreased expression of SIRT1 in hearts of old rats with emphysema and thus
might be a therapeutic modality for cardiac injury complicated in chronic
obstructive pulmonary disease (COPD). |
Which is the receptor for substrates of Chaperone Mediated Autophagy? | Chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective removal of damaged cytosolic proteins. The LAMP2A (Lysosome-associated membrane protein 2 isoform A) functions as a receptor for cytosolic proteins and also as essential component of the CMA translocation complex [28]. Cytosolic substrate proteins bind to monomers of LAMP-2A, which then multimerizes to form the complex required for substrate transmembrane import. | Intracellular protein degradation rates decrease with age in many tissues and
organs. In cultured cells, chaperone-mediated autophagy, which is responsible
for the selective degradation of cytosolic proteins in lysosomes, decreases with
age. In this work we use lysosomes isolated from rat liver to analyze
age-related changes in the levels and activities of the main components of
chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show
lower rates of chaperone-mediated autophagy, and both substrate binding to the
lysosomal membrane and transport into lysosomes decline with age. A progressive
age-related decrease in the levels of the lysosome-associated membrane protein
type 2a that acts as a receptor for chaperone-mediated autophagy was responsible
for decreased substrate binding in lysosomes from old rats as well as from late
passage human fibroblasts. The cytosolic levels and activity of the 73-kDa
heat-shock cognate protein required for substrate targeting to lysosomes were
unchanged with age. The levels of lysosome-associated hsc73 were increased only
in the oldest rats. This increase may be an attempt to compensate for reduced
activity of the pathway with age. Lamp2a acts as a receptor in the lysosomal membrane for substrate proteins of
chaperone-mediated autophagy. Using antibodies specific for the cytosolic tail
of lamp2a and others recognizing all lamp2 isoforms, we found that in rat liver
lamp2a represents 25% of lamp2s in the lysosome. We show that lamp2a levels in
the lysosomal membrane in rat liver and fibroblasts in culture directly
correlate with rates of chaperone-mediated autophagy in a variety of
physiological and pathological conditions. The concentration of other lamp2s in
the lysosomal membrane show no correlation under the same conditions.
Furthermore, substrate proteins bind to lamp2a but not to other lamp2s. Four
positively-charged amino acids uniquely present in the cytosolic tail of lamp2a
are required for the binding of substrate proteins. Lamp2a also distributes to
an unique subpopulation of perinuclear lysosomes in cultured fibroblasts in
response to serum withdrawal, and lamp2a, more than other lamp2s, tends to
multimerize. These characteristics may be important for lamp2a to act as a
receptor for chaperone-mediated autophagy. The selective degradation of cytosolic proteins in lysosomes by
chaperone-mediated autophagy depends, at least in part, on the levels of a
substrate receptor at the lysosomal membrane. We have previously identified this
receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed
that levels of lamp2a at the lysosomal membrane directly correlate with the
activity of the proteolytic pathway. Here we show that levels of lamp2a at the
lysosomal membrane are mainly controlled by changes in its half-life and its
distribution between the lysosomal membrane and the matrix. The lysosomal
degradation of lamp2a requires the combined action of at least two different
proteolytic activities at the lysosomal membrane. Lamp2a is released from the
membrane by the action of these proteases, and then the truncated lamp2a is
rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is
a regulated process that is inhibited in the presence of substrates for
chaperone-mediated autophagy and under conditions that activate that type of
autophagy. Uptake of substrate proteins also results in transport of some intact
lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can
be reinserted back into the lysosomal membrane. The traffic of lamp2a through
the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion
requires the lysosomal membrane potential and protein components of the
lysosomal membrane. The distribution of lamp2a between the lysosomal membrane
and matrix is a dynamic process that contributes to the regulation of lysosomal
membrane levels of lamp2a and consequently to the activity of the
chaperone-mediated autophagic pathway. Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of
unknown physiological function. We now demonstrate that this protease activity
triggers the degradation of the lysosome-associated membrane protein type 2a
(lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of
lamp2a is important because its level in the lysosomal membrane is a
rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a
degradation, higher levels of lamp2a and higher rates of CMA. Restoration of
PPCA protease activity increases rates of lamp2a degradation, reduces levels of
lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the
lysosomal membrane and cleaves lamp2a near the boundary between the luminal and
transmembrane domains. In addition to the well-studied role of PPCA in targeting
and protecting two lysosomal glycosidases, we have defined a role for the
proteolytic activity of this multifunctional protein. Chaperone-mediated autophagy (CMA) is a selective pathway for the degradation of
cytosolic proteins in lysosomes. CMA declines with age because of a decrease in
the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal
receptor for this pathway. We have selectively blocked the expression of LAMP-2A
in mouse fibroblasts in culture and analyzed the cellular consequences of
reduced CMA activity. CMA-defective cells maintain normal rates of long-lived
protein degradation by up-regulating macroautophagy, the major form of
autophagy. Constitutive up-regulation of macroautophagy is unable, however, to
compensate for all CMA functions. Thus, CMA-defective cells are more sensitive
to stressors, suggesting that, although protein turnover is maintained, the
selectivity of CMA is necessary as part of the cellular response to stress. Our
results also denote the existence of cross-talk among different forms of
autophagy. Parkinson disease (PD) is the most common neurodegenerative movement disorder.
An increase in the amount of alpha-synuclein protein could constitute a cause of
PD. Alpha-synuclein is degraded at least partly by chaperone-mediated autophagy
(CMA). The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is
associated with familial PD. However, the relationship between alpha-synuclein
and UCH-L1 in the pathogenesis of PD has remained largely unclear. In this
study, we found that UCH-L1 physically interacts with LAMP-2A, the lysosomal
receptor for CMA, and Hsc70 and Hsp90, which can function as components of the
CMA pathway. These interactions were abnormally enhanced by the I93M mutation
and were independent of the monoubiquitin binding of UCH-L1. In a cell-free
system, UCH-L1 directly interacted with the cytosolic region of LAMP-2A.
Expression of I93M UCH-L1 in cells induced the CMA inhibition-associated
increase in the amount of alpha-synuclein. Our findings may provide novel
insights into the molecular links between alpha-synuclein and UCH-L1 and suggest
that aberrant interaction of mutant UCH-L1 with CMA machinery, at least partly,
underlies the pathogenesis of PD associated with I93M UCH-L1. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed abundantly in neurons
and has been reported to be a major target of oxidative/carbonyl damage
associated with sporadic Parkinson's disease (PD). The I93M mutation in UCH-L1
is also associated with familial PD. We recently reported that UCH-L1 physically
interacts with LAMP-2A, the lysosomal receptor for chaperone-mediated autophagy
(CMA), and Hsc70 and Hsp90, both of which can function as components of the CMA
pathway. We found that the levels of these interactions were aberrantly
increased by the I93M mutation, and that expression of I93M UCH-L1 in cells
induced the CMA inhibition-associated increase in the amount of alpha-synuclein,
a risk factor for PD. The interactions of UCH-L1 with LAMP-2A, Hsc70 and Hsp90
were also abnormally enhanced by carbonyl modification of UCH-L1. We propose
that aberrant interactions of UCH-L1 variants with CMA machinery, at least
partly, underlie the pathogenesis of I93M UCH-L1-associated PD, and possibly of
sporadic PD. Our findings may provide novel insights into the links between
familial and sporadic PD. Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which
specific cytosolic proteins are sent to lysosomes for degradation. Substrate
proteins bind to the lysosomal membrane through the lysosome-associated membrane
protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene,
and this binding is limiting for their degradation via CMA. However, the
mechanisms of substrate binding and uptake remain unknown. We report here that
LAMP-2A organizes at the lysosomal membrane into protein complexes of different
sizes. The assembly and disassembly of these complexes are a very dynamic
process directly related to CMA activity. Substrate proteins only bind to
monomeric LAMP-2A, while the efficient translocation of substrates requires the
formation of a particular high-molecular-weight LAMP-2A complex. The two major
chaperones related to CMA, hsc70 and hsp90, play critical roles in the
functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we
have identified a novel function for hsc70 in the disassembly of LAMP-2A from
these complexes, whereas the presence of lysosome-associated hsp90 is essential
to preserve the stability of LAMP-2A at the lysosomal membrane. A subset of cytosolic proteins can be selectively degraded in lysosomes through
chaperone-mediated autophagy. The lysosomal-membrane protein type 2A (LAMP-2A)
acts as the receptor for the substrates of chaperone-mediated autophagy (CMA),
which should undergo unfolding before crossing the lysosomal membrane and
reaching the lumen for degradation. Translocation of substrates is assisted by
chaperones on both sides of the membrane, but the actual steps involved in this
process and the characteristics of the translocation complex were, for the most
part, unknown. We have now found that rather than a stable translocon at the
lysosomal membrane, CMA substrates bind to monomers of LAMP-2A driving the
organization of this protein into a high molecular weight multimeric complex
that mediates translocation. Assembly and disassembly of LAMP-2A into and from
this complex is dynamic and it is regulated by hsc70 and hsp90, the two
lysosomal chaperones related to CMA. This work thus unveils a unique mechanism
of protein translocation across the lysosomal membrane, which involves only
transient discontinuity of the membrane. The possible advantages of this
transitory lysosomal translocon are discussed in light of the unique properties
of the lysosomal compartment. Promoting the degradation of Hsp90 client proteins by inhibiting Hsp90, an
important protein chaperone, has been shown to be a promising new anticancer
strategy. In this study, we show that an oxazoline analogue of apratoxin A
(oz-apraA), a cyclodepsipeptide isolated from a marine cyanobacterium, promotes
the degradation of Hsp90 clients through chaperone-mediated autophagy (CMA). We
identify a KFERQ-like motif as a conserved pentapeptide sequence in the kinase
domain of epidermal growth factor receptor (EGFR) necessary for recognition as a
CMA substrate. Mutation of this motif prevents EGFR degradation by CMA and
promotes the degradation of EGFR through the proteasomal pathway in
oz-apraA-treated cells. Oz-apraA binds to Hsc70/Hsp70. We propose that apratoxin
A inhibits Hsp90 function by stabilizing the interaction of Hsp90 client
proteins with Hsc70/Hsp70 and thus prevents their interactions with Hsp90. Our
study provides the first examples for the ability of CMA to mediate degradation
of membrane receptors and cross talks of CMA and proteasomal degradation
mechanisms. Autophagy delivers cytoplasmic constituents for lysosomal degradation, and
thereby facilitates pathogen degradation and pathogen fragment loading onto MHC
molecules for antigen presentation to T cells. Herpesviruses have been used to
demonstrate these novel functions of autophagy, which previously has been
primarily appreciated for its pro-survival role during starvation. In this
review, we summarize recent findings how macroautophagy restricts herpesvirus
infections directly, how macroautophagy and chaperone mediated autophagy
contribute to herpesviral antigen presentation on MHC molecules, and which
mechanisms herpesviruses have developed to interfere with these pathways. These
studies suggest that herpesviruses significantly modulate autophagy to escape
from its functions in innate and adaptive immunity. Parkinson's disease (PD) is a progressive neurodegenerative disease caused by
interaction of genetic and environmental factors. To date, genetic genes and
variants causing PD remain largely unknown. Autophagy is a conserved cellular
process including three subtypes, macroautophagy (hereafter referred to as
autophagy), microautophagy and chaperone-mediated autophagy (CMA). Although
reduced CMA and induced autophagy are observed in human PD brain samples, cell
and animal PD models, CMA and autophagy have not been systemically studied in
sporadic PD patients. In the peripheral leukocytes of sporadic PD patients, we
examined gene expression levels of lysosome-associated membrane 2 (LAMP-2), a
CMA receptor and a limiting step, and microtubule-associated protein 1 light
chain 3 (LC3), product of which is sequentially cleaved and lipidated to form
LC3-II as an autophagosome marker. Compared to age- and sex-matched healthy
controls, LAMP-2 gene expression and protein levels in sporadic PD patients were
significantly decreased, which may lead to reduced CMA activity and impaired
fusion of autophagosome and lysosome. LC3 gene expression and LC3-II protein
levels were significantly increased in sporadic PD patients, suggesting that
autophagosomes are accumulated. Our findings, decreased LAMP-2 gene expression
and increased LC3 gene expression, are consistent to the previous studies with
dopaminergic neuronal cells in vitro and in vivo, which may contribute to the
pathogenesis of sporadic PD by altering CMA and autophagy activities. The
genetic causes leading to decreased LAMP-2 gene expression need further
investigation and genetic or pharmacological restoration of LAMP-2 might be a
novel strategy for treating PD patients. Nuclear receptor co-repressor (N-CoR) plays important role in transcriptional
control mediated by several tumor suppressor proteins. Recently, we reported a
role of misfolded-conformation dependent loss (MCDL) of N-CoR in the activation
of oncogenic survival pathway in acute promyelocytic leukemia (APL). Since N-CoR
plays important role in cellular homeostasis in various tissues, therefore, we
hypothesized that an APL like MCDL of N-CoR might also be involved in other
maligcy. Indeed, our initial screening of N-CoR status in various leukemia
and solid tumor cells revealed an APL like MCDL of N-CoR in primary and
secondary tumor cells derived from non-small cell lung cancer (NSCLC). The NSCLC
cell specific N-CoR loss could be blocked by Kaletra, a clinical grade protease
inhibitor and by genistein, an inhibitor of N-CoR misfolding previously
characterized by us. The misfolded N-CoR presented in NSCLC cells was linked to
the amplification of ER stress and was subjected to degradation by NSCLC cell
specific aberrant protease activity. In NSCLC cells, misfolded N-CoR was found
to be associated with Hsc70, a molecular chaperone involved in chaperone
mediated autophagy (CMA). Genetic and chemical inhibition of Lamp2A, a rate
limiting factor of CMA, significantly blocked the loss of N-CoR in NSCLC cells,
suggesting a crucial role of CMA in N-CoR degradation. These findings identify
an important role of CMA-induced degradation of misfolded N-CoR in the
neutralization of ER stress and suggest a possible role of misfolded N-CoR
protein in the activation of oncogenic survival pathway in NSCLC cells. RPE65-related Leber's congenital amaurosis (LCA) is a rod-cone dystrophy whose
clinical outcome is mainly attributed to the loss of rod photoreceptors followed
by cone degeneration. Pathogenesis in Rpe65(-/-) mice is characterized by a slow
and progressive degeneration of rods dependent on the constitutive activation of
unliganded opsin. We previously reported that this opsin-mediated apoptosis of
rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death
activity. In this study, we report early initial apoptosis in the newly
differentiated retina of Rpe65(-/-) mice. Apoptotic photoreceptors were
identified as rods and resulted from pathological phototransduction signaling.
This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic
activity, while activation of the caspases was not induced. Following cellular
stress, multiple signaling pathways are initiated which either commit cells to
death or trigger pro-survival responses including autophagy. We report that
Bcl-2-related early rod apoptosis was associated with the upregulation of
autophagy markers including chaperone-mediated autophagy (CMA) substrate
receptor LAMP-2 and lysosomal hydrolases Cathepsin S and Lysozyme. This suggests
that lysosomal-mediated autophagy may be triggered in response to early rod
apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-related
primary stress induces early signaling events, which trigger
Bax-induced-apoptotic pathway and autophagy-mediated cellular response. These
events may determine retinal cell fate, progression and severity of the disease. Cytosolic proteins can be selectively delivered to lysosomes for degradation
through a type of autophagy known as chaperone-mediated autophagy (CMA). CMA
contributes to intracellular quality control and to the cellular response to
stress. Compromised CMA has been described in aging and in different age-related
disorders. CMA substrates cross the lysosomal membrane through a translocation
complex; consequently, changes in the properties of the lysosomal membrane
should have a marked impact on CMA activity. In this work, we have analyzed the
impact that dietary intake of lipids has on CMA activity. We have found that
chronic exposure to a high-fat diet or acute exposure to a cholesterol-enriched
diet both have an inhibitory effect on CMA. Lysosomes from livers of
lipid-challenged mice had a marked decrease in the levels of the CMA receptor,
the lysosome-associated membrane protein type 2A, because of loss of its
stability at the lysosomal membrane. This accelerated degradation of
lysosome-associated membrane protein type 2A, also described as the mechanism
that determines the decline in CMA activity with age, results from its increased
mobilization to specific lipid regions at the lysosomal membrane. Comparative
lipidomic analyses revealed qualitative and quantitative changes in the lipid
composition of the lysosomal membrane of the lipid-challenged animals that
resemble those observed with age. Our findings identify a previously unknown
negative impact of high dietary lipid intake on CMA and underscore the
importance of diet composition on CMA malfunction in aging. BACKGROUND: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome
protein degradation pathway. The role of CMA in normal neuronal functions and in
neural disease pathogenesis remains unclear, in part because there is no
available method to monitor CMA activity at the single-cell level.
METHODOLOGY/PRINCIPAL FINDINGS: We sought to establish a single-cell monitoring
method by visualizing translocation of CMA substrates from the cytosol to
lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT
(GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured
cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated
HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA
and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for
CMA, and by treatments that inactivate CMA. These results suggest that lysosomal
accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed
that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA
activity in cultured PCs.
CONCLUSION/SIGNIFICANCE: In the present study, we established a novel
fluorescent-based method to evaluate CMA activity in a single neuron. This novel
method should be useful and valuable for evaluating the role of CMA in various
neuronal functions and neural disease pathogenesis. In neurodegenerative diseases, it remains unclear why certain brain regions are
selectively vulnerable to protein aggregation. In transgenic mice expressing
human A53T α-synuclein, the brainstem and spinal cord develop the most prominent
α-synuclein inclusions which correlate with age-dependent motor dysfunction.
Herein we present the novel finding that this selective aggregation is in part
dependent on the inability of chaperone-mediated autophagy (CMA) to effectively
degrade α-synuclein in these brain regions. Lysosomal assays revealed that CMA
activity was significantly decreased in aggregation-prone regions compared to
the remainder of the brain. Previously, CMA activity has been shown to be
proportional to levels of the CMA receptor Lamp-2a. Using antibodies, brain
tissue from Lamp-2a null mice, enzymatic deglycosylation, and mass spectrometry,
we identified Lamp2a as a novel 72kDa glycoprotein in the mouse brain.
Examination of Lamp-2a levels revealed differences in expression across brain
regions. The brainstem and the spinal cord had a more than three-fold greater
levels of Lamp-2a as compared to regions less vulnerable to aggregation and
exhibited a selective upregulation of Lamp-2a during development of α-synuclein
inclusions. Despite this dynamic response of Lamp-2a, the levels of substrates
bound to the brain lysosomes as well as the rates of substrate uptake and
degradation were not proportional to the levels of Lamp-2a. These regional
differences in CMA activity and Lamp-2a expression were found in both
non-transgenic mice as well as A53T α-syn mice. Therefore, these are inherent
variations and not a transgene-specific effect. However, differences in CMA
activity may render select brain regions vulnerable to homeostatic dysfunction
in the presence of stressors such as overexpression of human A53T α-syn.
Collectively, the data provide a potential mechanism to explain the dichotomy of
vulnerability or resistance that underlies brain regions during aggregate
formation in neurodegenerative disease. BACKGROUND: The family of lysosome-associated membrane proteins (LAMP) comprises
the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific
proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68).
LAMPs have been implicated in a multitude of cellular processes, including
phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a
receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal
Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of
the glycoconjugate coat present on the inside of the lysosomal membrane, the
'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain
of 150 to 200 amino acids with two disulfide bonds.
RESULTS: The crystal structure of the conserved domain of human DC-LAMP was
solved. It is the first high-resolution structure of a heavily glycosylated
lysosomal membrane protein. The structure represents a novel β-prism fold formed
by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible
loops and a hydrophobic pocket represent possible sites of molecular
interaction. Computational models of the glycosylated luminal regions of LAMP-1
and LAMP-2 indicate that the proteins adopt a compact conformation in close
proximity to the lysosomal membrane. The models correspond to the thickness of
the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron
microscopy.
CONCLUSION: The conserved luminal domain of lysosome-associated membrane
proteins forms a previously unknown β-prism fold. Insights into the structure of
the lysosomal glycoprotein coat were obtained by computational models of the
LAMP-1 and LAMP-2 luminal regions. A single cell has the potential to kill an entire human being. Efforts to cure
cancer are limited by survival of individual cancer cells despite immune
surveillance and toxic therapies. Understanding the intricate network of
pathways that maintain cellular homeostasis and mediate stress response or
default into cell death is critical to the development of strategies to
eradicate cancer. Autophagy, proteasomal degradation and the unfolded protein
response (UPR) are cellular pathways that degrade and recycle excess or damaged
proteins to maintain cellular homeostasis and survival. This review will discuss
autophagy and how it is integrated with proteasomal degradation and UPR to
govern cell fate through restoration of cellular homeostasis or default into the
apoptotic cell death pathway. The first response of autophagy is macroautophagy,
which sequesters cytoplasm including organelles inside double-membraned
autophagosome vesicles that fuse with lysosomes to degrade and recycle the
contents. Ubiquitination patterns on proteins targeted for degradation determine
whether adapter proteins will bring them to developing autophagosomes or to
proteasomes. Macroautophagy is followed by chaperone-mediated autophagy (CMA),
in which Hsc70 (Heat shock cognate 70) selectively binds proteins with exposed
KFERQ motifs and pushes them inside lysosomes through the LAMP-2A
(Lysosome-associated membrane protein type 2A) receptor. These two processes and
the lesser understood microautophagy, which involves direct engulfment of
proteins into lysosomes, occur at basal and induced levels. Insufficient
proteasome function or ER stress induction of UPR can induce autophagy, which
can mitigate damage and stress. If this network is incapable of repairing the
damage or overcoming continued stress, the default pathway of apoptosis is
engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics
has led to clinical trials investigating combinations of HCQ
(hydroxychloriquine) suppression of autophagy with apoptosis-inducing agents.
Further study of the complex integration of autophagy, proteasomal degradation,
UPR and apoptosis is likely to provide additional targets for our fight against
cancer. This article is part of a Special Issue entitled "Apoptosis: Four
Decades Later". AIMS: Chaperone-mediated autophagy (CMA) is a selective mechanism for the
degradation of soluble cytosolic proteins bearing the sequence KFERQ. These
proteins are targeted by chaperones and delivered to lysosomes where they are
translocated into the lysosomal lumen and degraded via the lysosome-associated
membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy
result in Danon disease characterized by hypertrophic cardiomyopathy. The
ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte
excitation-contraction and its dysfunction can lead to cardiac failure. Whether
RyR2 is degraded by CMA is unknown.
METHODS AND RESULTS: To induce CMA, cultured neonatal rat cardiomyocytes were
treated with geldanamycin (GA) to promote protein degradation through this
pathway. GA increased LAMP-2A levels together with its redistribution and
colocalization with Hsc70 in the perinuclear region, changes indicative of CMA
activation. The inhibition of lysosomes but not proteasomes prevented the loss
of RyR2. The recovery of RyR2 content after incubation with GA by siRNA
targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis
also revealed that the RyR2 sequence harbours six KFERQ motifs which are
required for the recognition Hsc70 and its degradation via CMA. Our data suggest
that presenilins are involved in RyR2 degradation by CMA.
CONCLUSION: These findings are consistent with a model in which oxidative damage
of the RyR2 targets it for turnover by presenilins and CMA, which could lead to
removal of damaged or leaky RyR2 channels. |
What are the hallmarks of congestive heart failure? | Congestive heart failure (HF) is a clinical syndrome, with hallmarks of fatigue and dyspnea, that continues to be highly prevalent and morbid. Common pathophysiologic features of HF include changes in left ventricle structure, function, and neurohormonal activation. Disturbed myocardial calcium handling is also one of the pathophysiologic hallmarks of congestive heart failure. One of the hallmarks of chronic congestive heart failure is an increase in sympathetic tone to the peripheral circulation and to the heart. It has been proposed that the activation of neurohormonal pathways and the formation of oxygen free radicals ultimately lead to the activation of a family of transcription factors that are involved in cardiac and vascular remodelling which are hallmarks of congestive heart failure. Myocardial failure ultimately leads to exaggerated neurohumoral compensatory mechanisms and derangements of the peripheral circulation, which are the hallmarks of congestive heart failure. Two additional hallmarks of this syndrome are sodium and water retention. Accumulation of oxidized matrix between the endothelium and cardiac muscle, and endocardial endothelial dysfunction, are also hallmarks of congestive heart failure. | Myocardial pump deficiency is regarded to be the hemodynamic hallmark of
congestive heart failure. A decline of arterial pressure in the systemic
circulation is counter-regulated by vasoconstriction in the arteriolar vascular
bed; the compensatory vasoconstriction, however, results in an increased
afterload that in turn aggravates myocardial pump deficiency. As part of the
counterregulatory systems the sympathetic nervous system is activated (increase
of neuronal activity, increased plasma norepinephrine) and the
renin-angiotensin-aldosterone system is stimulated as well (increased plasma
renin activity, elevated angiotensin II serum levels, hyperaldosteronism). In
parallel, serum levels of antidiuretic hormone (ADH) is despite a serum
hypoosmolarity increased and only poorly compensated by release of the atrial
natriuretic peptide. On the cellular level, congestive heart failure leads to a
shift of the expression of contractile proteins towards to fetal forms (for
instance myosin-isoenzymes). Although the counterregulatory activation of the
neuroendocrine systems vasoconstricts the peripheral arteries thereby
maintaining perfusion of vital organs, the rise in afterload ultimately leads to
a progression of congestive heart failure. Consequently, vasodilators (such as
ACE-inhibitors) that not only induce vasodilation in the peripheral arteries,
but also inhibit progressive neuroendocrine stimulation evolved as excellent
compounds for treating congestive heart failure. Chronic heart failure results from two processes, i.e., myocardial and
congestive failure. Myocardial failure is clinically silent, most often
progresses slowly, and is documented by a depressed left ventricular ejection
fraction. Multiple etiologic factors include systolic and diastolic overloads,
myocardial necrosis and/or ischemia, and, perhaps, microvascular spasm.
Myocardial failure ultimately leads to exaggerated neurohumoral compensatory
mechanisms and derangements of the peripheral circulation, which are the
hallmarks of congestive heart failure. At that stage of the syndrome, patients
have symptoms, initially, with exercise and, later, at rest. Objective
assessment of severity is afforded by determination of maximal oxygen uptake
during maximal exercise testing. When congestive heart failure supervenes, the
prognosis is poor. Current medical therapy is aimed at improving the
derangements of the peripheral circulation, which relieves the symptoms but
leaves the primary myocardial process unaffected. The goal of future therapy is
to intervene at an earlier stage of the syndrome to halt or even partially
reverse the myocardial failure. One of the hallmarks of chronic congestive heart failure is an increase in
sympathetic tone to the peripheral circulation and to the heart. A correlation
between plasma norepinephrine and the severity of the heart failure state has
been demonstrated. One mechanism that has been proposed to account for this
sympathoexcitation is a depression in the baroreflex and, perhaps, cardiac
reflex control of sympathetic nerve activity. This review summarizes work from
several laboratories, including our own, that documents a depressed baroreflex
control of heart rate and sympathetic nerve activity in both animals and humans
with heart failure. The mechanism of the depressed baroreflex most likely is
caused by reduced baroreceptor sensitivity as well as enhanced input to the
central nervous system from cardiac receptors that are chemosensitive. Although
sympathetic tone and arterial baroreflex sensitivity are altered in heart
failure, there have been no studies showing a cause-and-effect relationship.
Increases in plasma norepinephrine are similar in baroreceptor-denervated and
intact dogs paced into heart failure. This latter observation cells into
question the traditional concept of baroreceptor-mediated increases in
sympathetic tone in heart failure. Many pathophysiological processes are activated in patients with congestive
heart failure, and several of these have been implicated in the progression of
the disease. The most important processes to be activated in heart failure are
the neurohormonal systems, which include the renigiotensin system, the
sympathetic nervous system and the endothelin system. In addition to the
neurohormonal systems, the formation of reactive oxygen free radicals is
increased in congestive heart failure. It has been proposed that the activation
of neurohormonal pathways and the formation of oxygen free radicals ultimately
lead to the activation of a family of transcription factors that are involved in
cardiac and vascular remodelling which are hallmarks of congestive heart
failure. In addition, the formation of oxygen free radicals has been implicated
in the process of apoptosis, or programmed cell death, which may contribute to
the continued loss of myocardial cells resulting in progressive decreases in
left ventricular function, while at the same time contributing to the cardiac
remodelling process which subsequently creates a pro-arrhythmic environment in
the myocardium. Carvedilol is a novel multiple-action neurohormonal antagonist
that has been shown to be effective in the management of congestive heart
failure. Carvedilol also possesses a number of additional activities which may
inhibit many of the chronic pathophysiological processes that are involved in
the progression of congestive heart failure. PURPOSE: To explain key concepts involved in the development and management of
congestive heart failure (CHF). An overview of medications commonly used in the
treatment of CHF is also presented.
DATA SOURCES: Selected clinical articles, major research studies, and clinical
guidelines.
CONCLUSIONS: The prevalence of CHF continues to increase and in the United
States alone accounts for more than two million outpatient clinic visits per
year at a cost of more than $10 billion annually. With the growing number of
people over age 65, coupled with the advances in medical technology, primary
care providers are treating many more patients with CHF.
IMPLICATIONS FOR PRACTICE: A thorough understanding of the pathophysiology,
especially the compensatory neurohormonal systems and ventricular remodeling
that are the hallmarks of CHF, is essential for consistent application of the
principles of management of this complex condition. Multidisciplinary approaches
led by advanced practice nurses have excellent outcomes and improve the quality
of life of patients with CHF. Congestive heart failure (HF) is a clinical syndrome, with hallmarks of fatigue
and dyspnea, that continues to be highly prevalent and morbid. Because of the
growing burden of HF as the population ages, the need to develop new
pharmacological treatments and therapeutic interventions is of paramount
importance. Common pathophysiologic features of HF include changes in left
ventricle structure, function, and neurohormonal activation. The recapitulation
of the HF phenotype in large animal models can allow for the translation of
basic science discoveries into clinical therapies. Models of myocardial
infarction/ischemia, ischemic cardiomyopathy, ventricular pressure and volume
overload, and pacing-induced dilated cardiomyopathy have been created in dogs,
pigs, and sheep for the investigation of HF and potential therapies. Large
animal models recapitulating the clinical HF phenotype and translating basic
science to clinical applications have successfully traveled the journey from
bench to bedside. Undoubtedly, large animal models of HF will continue to play a
crucial role in the elucidation of biological pathways involved in HF and the
development and refinement of HF therapies. Disturbed myocardial calcium (Ca(+)) handling is one of the pathophysiologic
hallmarks of cardiovascular diseases such as congestive heart failure, cardiac
hypertrophy, and certain types of tachyarrhythmias. Pharmacologic treatment of
these diseases thus focuses on restoring myocardial Ca(2+) homeostasis by
interacting with Ca(2+)-dependent signaling pathways. In this article, we review
the currently used pharmacologic agents that are able to restore or maintain
myocardial Ca(2+) homeostasis and their mechanism of action as well as emerging
new substances. |
What is the mode of inheritance of short QT syndrome? | The short QT syndrome has an autosomal dominant mode of inheritance. | Sudden cardiac death in individuals with structurally normal hearts accounts for
approximately 20% of sudden cardiac death cases. Patients in this subgroup
suffer from what has been named "electrical diseases" which are gradually coming
into focus as inherited ion channelopathies, diseases of anchoring proteins or
of intracellular calcium regulating proteins. From 1993, the Short QT Syndrome
(SQTS) came to our attention, as a new inherited "electrical disease" associated
with increased risk of sudden cardiac death and atrial fibrillation. Mutations
of Ikr, Iks, Ikl channels cause dysfunctional Iks, Ikr, Ikl channels with an
increase in the net outward K current leading to shortening of repolarization.
This in turn leads to a shorter QT interval on the ECG and shorter atrial and
ventricular refractory periods with increased susceptibility to VF and AF. There
seems to be an autosomal domit mode of inheritance. The clinical profile of
SQTS consists of: family history of sudden cardiac death, personal history of
palpitations, syncope, dizziness, resuscitated SCD, history of AF and documented
VF. It is important to emphasize that SQTS is symptomatic from early age
(new-born) to old age. Therefore, it is possible that SQTS accounts for some of
the sudden infant death syndrome cases and for some cases of AF, especially lone
AF. The only efficient treatment for ventricular arrhythmias is ICD, associated
with drugs (Quinidine or Propaphenone) for AF prophylaxis and for reducing the
number of ventricular arrhythmic events (and ICD discharges). Conduction disorders result in cardiac arrhythmias that may be fatal.
Histiocytoid cardiomyopathy, Arrhythmogenic right ventricular dysplasia,
Isolated noncompaction of the left ventricle, Long QT syndrome (LQTS) and
Brugada syndrome, are all well described. Congenital short QT syndrome is a new
familial primary electrical disease of the heart, which is characterized by
abnormally short QT interval and paroxysmal atrial and ventricular
tachyarrhythmias, including sudden cardiac death. An autosomal domit mode of
inheritance has been suggested. Catecholaminergic polymorphic ventricular
tachycardia is an inherited disease and occurs in the absence of structural
heart disease or known associated syndromes. Although the histological
appearance of some of these disorders may be diagnostic, molecular analysis is
necessary to define clearly the particular type of cardiomyopathy. The past 2 decades have witnessed the emergence of many disease states related
to ion-channel disorders, the so-called "channelopathies," usually associated
with structurally normal hearts. The initial emphasis was directed toward the
congenital long QT syndrome and the Brugada syndrome. Recently, the hereditary
short QT syndrome has emerged as yet another rare channelopathy. This autosomal
domit syndrome can afflict infants, children, or young adults; often a
remarkable family background of sudden cardiac death is elucidated. The
electrocardiogram is characterized by a strikingly short QT interval (typically
<320 milliseconds); virtual absence of the ST segment; and tall, peaked,
narrow-based T waves. There is a marked propensity for paroxysmal atrial
fibrillation, and increased risk for sudden cardiac death from ventricular
tachyarrhythmias. At electrophysiology study, short atrial and ventricular
refractory periods are found, with easily inducible atrial fibrillation and
polymorphic ventricular tachycardia with programmed electrical stimulation.
Gain-of-function mutations in 3 genes encoding potassium channels have been
identified, which explain the abbreviated repolarization seen in this condition.
The suggested treatment is an implantable cardioverter-defibrillator, though the
possibilities of inappropriate shocks have caused some concern, especially in
younger patients. The ability of quinidine and disopyramide to prolong the QT
interval has the potential to be effective pharmacological therapy for patients
with short QT syndrome, but awaits additional confirmatory clinical data. |
List clinical trials that have directly compared microsurgical clipping with endovascular coiling for treatment of ruptured brain aneurysms? | Barrow Ruptured Aneurysm Trial (BRAT) and international subarachnoid aneurysmal trial (ISAT) have directly compared microsurgical clipping with endovascular coiling for treatment of ruptured brain aneurysms. FIAT study, a clinical care trial aiming to compare angiographic and clinical outcomes following treatment with a Flow-Diverter or with the best conventional treatment option (including clipping) is underway. | BACKGROUND: Two types of treatment are being used for patients with ruptured
intracranial aneurysms: endovascular detachable-coil treatment or craniotomy and
clipping. We undertook a randomised, multicentre trial to compare these
treatments in patients who were suitable for either treatment because the
relative safety and efficacy of these approaches had not been established. Here
we present clinical outcomes 1 year after treatment.
METHODS: 2143 patients with ruptured intracranial aneurysms, who were admitted
to 42 neurosurgical centres, mainly in the UK and Europe, took part in the
trial. They were randomly assigned to neurosurgical clipping (n=1070) or
endovascular coiling (n=1073). The primary outcome was death or dependence at 1
year (defined by a modified Rankin scale of 3-6). Secondary outcomes included
rebleeding from the treated aneurysm and risk of seizures. Long-term follow up
continues. Analysis was in accordance with the randomised treatment.
FINDINGS: We report the 1-year outcomes for 1063 of 1073 patients allocated to
endovascular treatment, and 1055 of 1070 patients allocated to neurosurgical
treatment. 250 (23.5%) of 1063 patients allocated to endovascular treatment were
dead or dependent at 1 year, compared with 326 (30.9%) of 1055 patients
allocated to neurosurgery, an absolute risk reduction of 7.4% (95% CI 3.6-11.2,
p=0.0001). The early survival advantage was maintained for up to 7 years and was
significant (log rank p=0.03). The risk of epilepsy was substantially lower in
patients allocated to endovascular treatment, but the risk of late rebleeding
was higher.
INTERPRETATION: In patients with ruptured intracranial aneurysms suitable for
both treatments, endovascular coiling is more likely to result in independent
survival at 1 year than neurosurgical clipping; the survival benefit continues
for at least 7 years. The risk of late rebleeding is low, but is more common
after endovascular coiling than after neurosurgical clipping. Cerebral aneurysms are treated by two methods: direct microsurgical clipping and
endovascular coiling. Both are selected based on definite guidelines for
clinicoradiological criteria as follows: Endovascular therapy comprising of GDC
embolization, CSF wash-out with UK or TP A were performed in cases with Hunt and
Kosnik grade 4 (GCS 7, 8), and grade 5 (without hydrocephalus or intracranial
hemorrhage), age>70 years, subacute stage (4--14 days of vasospasm), basilar
aneurysm and peripheral MCA/PCA aneurysms. Microsurgical clipping with a
drainage procedure was performed in cases with Hunt and Kosnik grades 0--3,
grade 4 (GCS 9--12), age less than 70 years, grade 5 with hydrocephalus or
intracerebral hematoma and acute stage (0--3 days after bleed). The patient's
outcome was measured using GOS (Glasgow outcome score) at the time of discharge.
In our series of severe (poor grade) SAH cases, 120 cases underwent clipping and
59 cases underwent coiling. Although they accounted for 37.8 % and 48 % of total
SAH cases, respectively, the outcome was satisfactory. Good recovery and
moderate disability, together termed "favorable outcome" was found in 69.16 % of
clipping cases and 44.06 % of coiling cases. Clipping had a better outcome than
coiling in cases of acute severe SAH in our series. The golden hour
resuscitation, pre-hospital care and the adjunctive treatment strategies like
hypothermia are discussed. A critical appraisal of the ISAT of microsurgical
clipping versus coiling is used for comparison of our results. DOI: 10.2147/vhrm.2007.03.03.243
PMCID: PMC2293965
PMID: 17703631 [Indexed for MEDLINE] BACKGROUND: Our aim was to assess the long-term risks of death, disability, and
rebleeding in patients randomly assigned to clipping or endovascular coiling
after rupture of an intracranial aneurysm in the follow-up of the International
Subarachnoid Aneurysm Trial (ISAT).
METHODS: 2143 patients with ruptured intracranial aneurysms were enrolled
between 1994 and 2002 at 43 neurosurgical centres and randomly assigned to
clipping or coiling. Clinical outcomes at 1 year have been previously reported.
All UK and some non-UK centres continued long-term follow-up of 2004 patients
enrolled in the original cohort. Annual follow-up has been done for a minimum of
6 years and a maximum of 14 years (mean follow-up 9 years). All deaths and
rebleeding events were recorded. Analysis of rebleeding was by allocation and by
treatment received. ISAT is registered, number ISRCTN49866681.
FINDINGS: 24 rebleeds had occurred more than 1 year after treatment. Of these,
13 were from the treated aneurysm (ten in the coiling group and three in the
clipping group; log rank p=0.06 by intention-to-treat analysis). There were 8447
person-years of follow-up in the coiling group and 8177 person-years of
follow-up in the clipping group. Four rebleeds occurred from a pre-existing
aneurysm and six from new aneurysms. At 5 years, 11% (112 of 1046) of the
patients in the endovascular group and 14% (144 of 1041) of the patients in the
neurosurgical group had died (log-rank p=0.03). The risk of death at 5 years was
significantly lower in the coiling group than in the clipping group (relative
risk 0.77, 95% CI 0.61-0.98; p=0.03), but the proportion of survivors at 5 years
who were independent did not differ between the two groups: endovascular 83%
(626 of 755) and neurosurgical 82% (584 of 713). The standardised mortality
rate, conditional on survival at 1 year, was increased for patients treated for
ruptured aneurysms compared with the general population (1.57, 95% CI 1.32-1.82;
p<0.0001).
INTERPRETATION: There was an increased risk of recurrent bleeding from a coiled
aneurysm compared with a clipped aneurysm, but the risks were small. The risk of
death at 5 years was significantly lower in the coiled group than it was in the
clipped group. The standardised mortality rate for patients treated for ruptured
aneurysms was increased compared with the general population.
FUNDING: UK Medical Research Council. Intracranial aneurysms, particularly large and giant, fusiform or recurrent
aneurysms are increasingly treated with flow diverters (FDs), a recently
introduced and approved neurovascular device. While some rare cases may not be
treated any other way, in most patients a more conventional, conservative, or
validated approach such as coiling, parent vessel occlusion, or surgical
clipping exists. Only a randomized clinical trial can answer the question of
which treatment option leads to better patient outcomes.We report the design of
the FIAT study, a clinical care trial aiming to compare angiographic and
clinical outcomes following treatment with a Flow-Diverter or with the best
conventional treatment option. The FIAT study will include both a randomized and
a registry portion. Patients will be proposed randomization to either FD
stenting or best conventional treatment option (observation, coiling, stenting,
or clipping) as determined by the treating physician. FIAT will recruit a total
of 338 patients, to show that i) FD stenting can be performed with an
'acceptable' immediate complication rate of less than 15% morbidity and
mortality (defined as mRS > 2); ii) FD stenting can increase from 75 to 90% the
proportion of patients with a "good outcome", defined as complete or
near-complete occlusion of the aneurysm AND a good clinical outcome (mRS ≥ 2) at
one year, as compared to the best conventional option. The FIAT study provides a
scientific and ethical context to care for patients eligible for flow-diversion
therapy. OBJECTIVE: In recent years the endovascular treatment of intracranial aneurysms
(coiling) has progressively gained recognition, particularly after the
publication of the International Subarachnoid Aneurysm Trial (ISAT) in 2002.
Despite the fact that in ISAT middle cerebral artery (MCA) aneurysms were
clearly underrepresented, the study is often used as an argument to favor
coiling above surgery in MCA aneurysms. Taken into account that MCA aneurysms
are very well accessible for surgery, a contemporary assessment of the benefits
of a preferred surgical strategy for MCA aneurysms was performed in a tertiary
neurovascular referral center.
METHODS: A prospectively kept single-center database of 151 consecutive patients
with an MCA aneurysm was reviewed over a 6-year period (2001-2006). Long-term
follow-up after surgical treatment of a ruptured MCA aneurysm was obtained in 74
out of 77 (96%) patients. The outcome was compared with relevant series in the
literature.
RESULTS: After a mean follow-up of 4.7 years, 59 out of 74 surgically treated
patients (80%) with a ruptured MCA aneurysm had a good outcome (mRankin 0-2).
All patients with an unruptured MCA aneurysm also had a good outcome after
clipping. This is well-matched with the findings of the literature search, and
competitive with the endovascular results.
CONCLUSION: Surgical clipping is recommended as the principal treatment strategy
for MCA aneurysms. This is not only ethically defendable in view of the surgical
results but also in line with a strategy to maintain surgical experience within
centralized neurovascular centers. Delayed cerebral ischemia (DCI) is an important cause of poor outcome after
aneurysmal subarachnoid hemorrhage (SAH). We studied differences in incidence
and impact of DCI as defined clinically after coiling and after clipping in the
International Subarachnoid Aneurysm Trial. We calculated odds ratios (OR) for
DCI for clipping versus coiling with logistic regression analysis. With coiled
patients without DCI as the reference group, we calculated ORs for poor outcome
at 2 months and 1 year for coiled patients with DCI and for clipped patients
without, and with DCI. With these ORs, we calculated relative excess risk due to
Interaction (RERI). Clipping increased the risk of DCI compared to coiling in
the 2,143 patients OR 1.24, 95% confidence interval (95% CI 1.01-1.51). Coiled
patients with DCI, clipped patients without DCI, and clipped patients with DCI
all had higher risks of poor outcome than coiled patients without DCI. Clipping
and DCI showed no interaction for poor outcome at 2 months: RERI 0.12 (95% CI
-1.16 to 1.40) or 1 year: RERI -0.48 (95% CI -1.69 to 0.74). Only for patients
treated within 4 days, coiling and DCI was associated with a poorer outcome at
1 year than clipping and DCI (RERI -2.02, 95% CI -3.97 to -0.08). DCI was more
common after clipping than after coiling in SAH patients in ISAT. Impact of DCI
on poor outcome did not differ between clipped and coiled patients, except for
patients treated within 4 days, in whom DCI resulted more often in poor outcome
after coiling than after clipping. OBJECTIVE: Ruptured vertebrobasilar (VB) saccular aneurysm is a difficult lesion
to treat, and is associated with high rates of morbidity and mortality. The aim
of this study is to investigate the risk factors associated with the clinical
outcome of ruptured VB aneurysms.
METHODS: A retrospective review of 29 patients with ruptured VB saccular
aneurysms between 2002 and 2010 was conducted between Jan 2002 and Dec 2010.
Univariate and multivariate analyses were performed for determination of the
statistical significance of the Glasgow Outcome Scale (GOS) at three months,
according to age, initial Hunt-Hess grade, the presence of acute hydrocephalus,
and treatment modality.
RESULTS: The study included 24 (82.7%) females and five (17.3%) males, with a
mean age of 59 years (range, 22-78 years). Seventeen patients were treated with
surgical clipping and 12 patients were treated with endovascular coil
embolization. No statistical significance was observed between clinical outcome
and treatment modalities (clipping or coiling; p = 0.803). Seventeen (58.6%)
patients achieved favorable outcome, defined as GOS score of 4-5, at 3 months.
Procedure-related complications occurred in seven patients (24.1%). Results of
multivariate analysis indicated that initial Hunt-Hess grade and the presence of
acute hydrocephalus were independent predictors of unfavorable outcome, defined
as GOS score of 1-3 (Odds ratio (OR) = 8.63, Confidence interval (CI) [95%]
1.11-66.84, p = 0.039 and OR = 36.64, CI [95%] 2.23-599.54, p = 0.012,
respectively).
CONCLUSION: The present study suggests that the clinical outcomes are related to
the initial Hunt-Hess grade and the presence of acute hydrocephalus in ruptured
saccular VB aneurysms. BACKGROUND: The use of stent-assisted coiling (SAC) has been shown to be a
treatment option for complex aneurysms. We reviewed systematically the immediate
and mid-term angiographic results following treatment of wide-necked aneurysms
with self-expanding stents and coils, as well as the peri- and postprocedural
rate of complications.
METHODS: A computerized database search was conducted from 01/2000 to 04/2011
using appropriate indexed terms on Pubmed. Inclusion criteria were: (1)
homogeneous populations of ≥10 patients with wide-necked aneurysms; (2) use of a
self-expandable neurovascular stent and coils for aneurysm treatment; (3)
immediate and follow-up angiographic results; and (4) periprocedural and delayed
thrombotic complications.
RESULTS: Seventeen studies were included, containing retrospectively collected
data on 656 patients/702 aneurysms. The target aneurysm was located on the
anterior circulation in 78.4% of patients. The immediate rate of complete
occlusion was 46.3%, (19.3-98.1%). The intra- and postprocedural rate of
intrastent thrombosis or thromboembolic event was 4.6% and 4.3%, respectively.
Complete occlusion was documented in 71.9% at last angiographic follow-up. The
rate of recanalization was 13.2% of aneurysms (0-28.8%). Delayed in-stent
stenosis occurred in 5.3% cases (0-20.6%).
CONCLUSION: SAC has been considered a treatment option for selected wide-necked
aneurysms in some institutions. The use of intracranial stents should take into
consideration the risk of ischemic complications, recanalization, delayed
in-stent stenosis; and the currently unknown lifetime risks for stenosis,
vascular injury, device failure, and aneurysm recurrence related to intracranial
stenting. There is an evident need for a prospective multicenter registry for
all treated patients with SAC. OBJECT: The authors report the 3-year results of the Barrow Ruptured Aneurysm
Trial (BRAT). The objective of this ongoing randomized trial is to compare the
safety and efficacy of microsurgical clip occlusion and endovascular coil
embolization for the treatment of acutely ruptured cerebral aneurysms and to
compare functional outcomes based on clinical and angiographic data. The 1-year
results have been previously reported.
METHODS: Two-hundred thirty-eight patients were assigned to clip occlusion and
233 to coil embolization. There were no anatomical exclusions. Crossovers were
allowed based on the treating physician's determination, but primary outcome
analysis was based on the initial assignment to treatment modality. Patient
outcomes were assessed independently using the modified Rankin Scale (mRS). A
poor outcome was defined as an mRS score>2. At 3 years' follow-up 349 patients
who had actually undergone treatment were available for evaluation. Of the 170
patients who had been originally assigned to coiling, 64 (38%) crossed over to
clipping, whereas 4 (2%) of 179 patients assigned to surgery crossed over to
clipping.
RESULTS: The risk of a poor outcome in patients assigned to clipping compared
with those assigned to coiling (35.8% vs 30%) had decreased from that observed
at 1 year and was no longer significant (OR 1.30, 95% CI 0.83-2.04, p=0.25). In
addition, the degree of aneurysm obliteration (p=0.0001), rate of aneurysm
recurrence (p=0.01), and rate of retreatment (p=0.01) were significantly better
in the group treated with clipping compared with the group treated with coiling.
When outcomes were analyzed based on aneurysm location (anterior circulation,
n=339; posterior circulation, n=69), there was no significant difference in the
outcomes of anterior circulation aneurysms between the 2 assigned groups across
time points (at discharge, 6 months, 1 year, or 3 years after treatment). The
outcomes of posterior circulation aneurysms were significantly better in the
coil group than in the clip group after the 1st year of follow-up, and this
difference persisted after 3 years of follow-up. However, while aneurysms in the
anterior circulation were well matched in their anatomical location between the
2 treatment arms, this was not the case in the posterior circulation where, for
example, 18 of 21 posterior inferior cerebellar artery aneurysms were in the
clip group.
CONCLUSIONS: Based on mRS scores at 3 years, the outcomes of all patients
assigned to coil embolization showed a favorable 5.8% absolute difference
compared with outcomes of those assigned to clip occlusion, although this
difference did not reach statistical significance (p=0.25). Patients in the clip
group had a significantly higher degree of aneurysm obliteration and a
significantly lower rate of recurrence and retreatment. In post hoc analysis
examining only anterior circulation aneurysms, no outcome difference between the
2 treatment cohorts was observed at any recorded time point. CLINICAL TRIAL
REGISTRATION NO.: NCT01593267 ( ClinicalTrials.gov ). |
What is the reason for the narcolepsy cases developed after H1N1 influenza vaccination? | The proposed mechanism for postvaccination narcolepsy is one in which an environmental trigger causes or enhances an antibody-mediated autoimmune response in patients with a preexisting genetic susceptibility. | INTRODUCTION: It has been suggested that the H1N1 vaccine may be a trigger for
the onset of narcolepsy-cataplexy, a rare disease whose autoimmune origin is
suspected.
OBSERVATIONS: We report two patients (a 9-year-old boy and an 18-year-old man)
with severe narcolepsy-cataplexy, in whom the illness appeared within 3-4 weeks
after H1N1 vaccination. In both cases, symptoms developed unusually abruptly and
they presented with severe daytime sleepiness and multiple daily cataplexy
attacks. Other similar cases have been recently reported associated with
H1N1 vaccine.
CONCLUSION: Although no formal link can be established, the unusual
characteristics of the reported cases and the striking temporal relationship
suggests that narcolepsy may be the result of an autoimmune reaction triggered
by H1N1 vaccination in susceptible individuals. The loss of hypothalamic hypocretin/orexin (hcrt) producing neurons causes
narcolepsy with cataplexy. An autoimmune basis for the disease has long been
suspected and recent results have greatly strengthened this hypothesis.
Narcolepsy with hcrt deficiency is now known to be associated with a Human
Leukocyte Antigen (HLA) and T-cell receptor (TCR) polymorphisms, suggesting that
an autoimmune process targets a single peptide unique to hcrt-cells via specific
HLA-peptide-TCR interactions. Recent data have shown a robust seasonality of
disease onset in children and associations with Streptococcus Pyogenes, and
influenza A H1N1-infection and H1N1-vaccination, pointing towards processes such
as molecular mimicry or bystander activation as crucial for disease development.
We speculate that upper airway infections may be common precipitants of a whole
host of CNS autoimmune complications including narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness, with or without
cataplexy. Associated features include sleep paralysis, hypnagogic or
hypnopompic hallucinations, and disturbed nocturnal sleep. Narcolepsy is
strongly associated with the HLA DQB1*0602 allele, and its symptoms stem from
destruction of hypocretin-secreting neurons in the hypothalamus. Recently
identified autoantibodies to Tribbles homologue 2 in some patients, as well as
cases associated with H1N1 vaccination, support an autoimmune mechanism. There
are many challenges in diagnosing and treating pediatric narcolepsy. Caution
must also be used in interpreting polysomnography and multiple sleep latency
test results in children. HLA testing is nonspecific, and no commercial test
exists to measure cerebrospinal fluid hypocretin levels. Neuroimaging has not
yet proven useful in primary narcolepsy. Treatment of sleepiness and cataplexy
in children requires extrapolating from adult studies. Hopefully, further
insights into the pathophysiology of narcolepsy will allow for new therapeutics
to manage the symptoms and modify the course of the disease. OBJECTIVES: To assess the incidence of narcolepsy between January 2000 and
December 2010 in children in western Sweden and its relationship to the
Pandemrix vaccination, and to compare the clinical and laboratory features of
these children.
METHODS: The children were identified from all local and regional pediatric
hospitals, child rehabilitation centers, outpatient pediatric clinics, and
regional departments of neurophysiology. Data collection was performed with the
aid of a standardized data collection form, from medical records and telephone
interviews with patients and parents. The laboratory and investigational data
were carefully scrutinized.
RESULTS: We identified 37 children with narcolepsy. Nine of them had onset of
symptoms before the H1N1 vaccination and 28 had onset of symptoms in
relationship to the vaccination. The median age at onset was 10 years. All
patients in the postvaccination group were positive for human leukocyte antigen
(HLA)-DQB1*0602. Nineteen patients in the postvaccination group, compared with
one in the prevaccination group, had a clinical onset that could be dated within
12 weeks.
CONCLUSION: Pandemrix vaccination is a precipitating factor for narcolepsy,
especially in combination with HLA-DQB1*0602. The incidence of narcolepsy was 25
times higher after the vaccination compared with the time period before. The
children in the postvaccination group had a lower age at onset and a more sudden
onset than that generally seen. An increased incidence of narcolepsy in children was detected in Scandinavian
countries where pandemic H1N1 influenza ASO3-adjuvanted vaccine was used. A
campaign of vaccination against pandemic H1N1 influenza was implemented in
France using both ASO3-adjuvanted and non-adjuvanted vaccines. As part of a
study considering all-type narcolepsy, we investigated the association between
H1N1 vaccination and narcolepsy with cataplexy in children and adults compared
with matched controls; and compared the phenotype of narcolepsy with cataplexy
according to exposure to the H1N1 vaccination. Patients with
narcolepsy-cataplexy were included from 14 expert centres in France. Date of
diagnosis constituted the index date. Validation of cases was performed by
independent experts using the Brighton collaboration criteria. Up to four
controls were individually matched to cases according to age, gender and
geographic location. A structured telephone interview was performed to collect
information on medical history, past infections and vaccinations. Eighty-five
cases with narcolepsy-cataplexy were included; 23 being further excluded
regarding eligibility criteria. Of the 62 eligible cases, 59 (64% males, 57.6%
children) could be matched with 135 control subjects. H1N1 vaccination was
associated with narcolepsy-cataplexy with an odds ratio of 6.5 (2.1-19.9) in
subjects aged<18 years, and 4.7 (1.6-13.9) in those aged 18 and over.
Sensitivity analyses considering date of referral for diagnosis or the date of
onset of symptoms as the index date gave similar results, as did analyses
focusing only on exposure to ASO3-adjuvanted vaccine. Slight differences were
found when comparing cases with narcolepsy-cataplexy exposed to H1N1 vaccination
(n=32; mostly AS03-adjuvanted vaccine, n=28) to non-exposed cases (n=30),
including shorter delay of diagnosis and a higher number of sleep onset rapid
eye movement periods for exposed cases. No difference was found regarding
history of infections. In this sub-analysis, H1N1 vaccination was strongly
associated with an increased risk of narcolepsy-cataplexy in both children and
adults in France. Even if, as in every observational study, the possibility that
some biases participated in the association cannot be completely ruled out, the
associations appeared robust to sensitivity analyses, and a specific analysis
focusing on ASO3-adjuvanted vaccine found similar increase. PURPOSE OF REVIEW: A number of European countries have reported a dramatic
increase in the rates of childhood narcolepsy with cataplexy in children
immunized with a split-virion adjuvanted swine flu vaccine. Here, we review the
strengths and weaknesses of these epidemiological studies and possible
neuroimmunological mechanisms.
RECENT FINDINGS: Initial concerns of a 13-fold increased relative risk of
narcolepsy were raised by the Scandinavian health protection agencies in 2010.
Subsequent retrospective studies support these findings in Canada, France,
Ireland, England and Denmark. The cases are predomitly young children who
present with severe and rapid onset of cataplexy as well as narcolepsy often
within a few weeks of vaccination. The proposed mechanism for postvaccination
narcolepsy is one in which an environmental trigger causes or enhances an
antibody-mediated autoimmune response in patients with a preexisting genetic
susceptibility. However, there have not yet been any reports of specific
autoimmunity, either antibody or T-cell-mediated.
SUMMARY: There is a strong association between narcolepsy and H1N1 vaccination.
However, whether this reflects a true increase in affected individuals or a
hastening of disease onset in individuals who would otherwise have developed
narcolepsy later will become clear in the coming years. The pathological
explanation of this association and narcolepsy is likely to be autoimmune,
although supportive evidence is lacking.Video abstract available: See the Video
Supplementary Digital Content 1 (http://links.lww.com/COPM/A9). |
Which are the biotracers used for detection of Alzheimer's disease using PET? | Pittsburgh compound B (PIB) was the first radiotracer capable of highlighting deposits of beta-amyloid—one pathological hallmark of Alzheimer's disease—in living individuals during a PET scan. The Alzheimer's Association helped fund early PIB development. The Association in 2006 also awarded a $2.1 million grant to the Alzheimer's Disease Neuroimaging Initiative (ADNI) to expand this long-term, nationwide study to include PIB-PET imaging.
18F flutemetamol (flute), another radiotracer that highlights beta-amyloid in a PET scan, is structurally identical to PIB except for one fluorine atom in place of a carbon atom. That small chemical change enables flutemetamol to remain stable significantly longer than does PIB, potentially increasing its usefulness outside research settings. In phase II study results reported in the Annals of Neurology, flutemetamol performed similarly to PIB. Additional testing is under way.
Florbetapir F 18 (18F-AV-45) is also a radiotracer that highlights brain beta-amyloid during a PET scan. At the 2010 Alzheimer's Association International Conference on Alzheimer's Disease (AAICAD), florbetapir's developer first reported data, later published in the JAMA, showing nearly perfect correlation between brain amyloid levels detected by florbetapir PET scans in study volunteers and levels found in autopsies of the same individuals a few months later. The developer has sought Food and Drug Administration (FDA) approval to market florbetapir under the brand name Amyvid. The FDA has said it will withhold approval until the developer establishes a professional training program to ensure accuracy and consistency in reading and interpreting Amyvid scans.
Florbetaben (BAY 94-9172) is another radiotracer designed to detect beta-amyloid during a PET scan. Phase II study results and other florbetaben data were reported at the 2010 Alzheimer's Association International Conference on Alzheimer's Disease (AAICAD). Phase II data were also later published in Lancet Neurology. Further studies are now under way. | Conflict of interest statement: Competing Interests: The author has declared
that no competing interests exist. Alzheimer's disease (AD) is difficult to diagnose in its early stages, and even
if detected early, there is no preventative treatment. Imaging modalities such
as MRI, PET, and SPECT have the potential to contribute to both the diagnosis of
Alzheimer's disease, as well as assist in the search for more effective
treatments. A number of AD-related biomarkers have been proposed and evaluated.
The use of PET imaging to detect alterations in regional brain metabolism using
[(18)F]FDG has enabled more sensitive and accurate early diagnosis of AD,
especially in conjunction with traditional medical evaluation. Additionally,
magnetic resoce imaging and spectroscopy provide a wide range of biomarkers
that have been shown to correlate with the progression of AD. Some of these
markers have been pursued in clinical trials. Progress has been made toward the
evaluation of other more AD-specific biomarkers. However, many questions remain
concerning the validity and sensitivity of these imaging biomarkers to aid in
the assessment of potential new treatments, especially those related to
increased levels of amyloid peptides in the brain. BACKGROUND: Amyloid senile plaques and tau neurofibrillary tangles are
neuropathological hallmarks of Alzheimer's disease that accumulate in the
cortical regions of the brain in persons with mild cognitive impairment who are
at risk for Alzheimer's disease. Noninvasive methods to detect these abnormal
proteins are potentially useful in developing surrogate markers for drug
discovery and diagnostics.
METHODS: We enrolled 83 volunteers with self-reported memory problems who had
undergone neurologic and psychiatric evaluation and positron-emission tomography
(PET). On the basis of cognitive testing, 25 volunteers were classified as
having Alzheimer's disease, 28 as having mild cognitive impairment, and 30 as
having no cognitive impairment (healthy controls). PET was performed after
injection of
2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile
(FDDNP), a molecule that binds to plaques and tangles in vitro. All subjects
also underwent 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) PET, and 72 underwent
magnetic resoce imaging (MRI).
RESULTS: Global values for FDDNP-PET binding (average of the values for the
temporal, parietal, posterior cingulate, and frontal regions) were lower in the
control group than in the group with mild cognitive impairment (P<0.001), and
the values for binding in the group with mild cognitive impairment were lower
than in the group with Alzheimer's disease (P<0.001). FDDNP-PET binding
differentiated among the diagnostic groups better than did metabolism on FDG-PET
or volume on MRI.
CONCLUSIONS: FDDNP-PET scanning can differentiate persons with mild cognitive
impairment from those with Alzheimer's disease and those with no cognitive
impairment. This technique is potentially useful as a noninvasive method to
determine regional cerebral patterns of amyloid plaques and tau neurofibrillary
tangles. Neuronal nicotinic acetylcholine receptors (nAChRs) are critical for higher
order cognitive processes. Post-mortem studies suggest reductions in nAChRs
(particularly the alpha(4)beta(2) subtype) with ageing and in Alzheimer's
disease (AD). This study aimed to; (1) quantify nAChR distribution in vivo with
2-[18F]fluoro-A-85380 (2-FA) in 15 early AD patients compared to 14 age-matched,
healthy controls (HC) and (2) correlate nAChR distribution with cognitive
performance in both groups. All participants were non-smokers and underwent
cognitive testing along with a dynamic PET scan after injection of 200 MBq of
2-FA. Brain regional 2-FA binding was assessed through a simplified estimation
of Distribution Volume (DV(S)). The AD group differed significantly from HC on
all cognitive measures employed, with impairments on measures of attention,
working memory, language, executive function, visuospatial ability, verbal
learning and verbal memory (p<.05). Contrary to post-mortem data this study
found no evidence of in vivo nAChR loss in early AD despite significant
cognitive impairment. Furthermore, no correlation between nAChR and cognitive
performance was found for either group. The findings of the current study
suggest preservation of nAChRs early in AD supporting previous studies. It is
possible that while the clinical 2-FA PET method described here may be
insensitive in detecting changes in early AD, such changes may be detected in
more advanced stages of the illness. OBJECTIVE: Adenosine is an endogenous modulator of synaptic functions in the
central nervous system. The effects of adenosine are mediated by at least four
adenosine receptor subtypes. Decreased density of adenosine A1 receptors, which
is a major subtype adenosine receptor in the hippocampus, has been reported in
vitro in Alzheimer's disease. We evaluated adenosine A1 receptor in the brain of
elderly normal subjects and patients with Alzheimer's disease (n = 8 and 6,
respectively), using positron emission tomography (PET) and 8-
dicyclopropylmethyl-1-[(11)C]methyl-3-propylxanthine ([(11)C]MPDX).
METHODS: A 60-min PET scan with [(11)C]MPDX was performed. The patients with
Alzheimer's disease also underwent PET with [(18)F]fluorodeoxyglucose (FDG). The
binding potential of [11C]MPDX was quantitatively calculated in the regions of
interest (ROIs) placed on the frontal, medial frontal, temporal, medial
temporal, parietal, and occipital cortices, striatum, thalamus, cerebellum, and
pons. Statistical parametric mapping (SPM2) was used for analysis of [(11)C]MPDX
and FDG-PET.
RESULTS: In the ROI-based analysis, the binding potential of [(11)C]MPDX in
patients with Alzheimer's disease was significantly lower in the temporal and
medial temporal cortices and thalamus than that in elderly normal subjects (P =
0.038, 0.028, and 0.039, respectively). SPM analysis also showed significant
decreased binding potential in the temporal and medial temporal cortices and
thalamus in patients with Alzheimer's disease. FDG uptake was significantly
decreased in the temporoparietal cortex and posterior cingulate gyrus.
CONCLUSIONS: Decreased binding of [(11)C]MPDX in patients with Alzheimer's
disease was detected in temporal and medial temporal cortices and thalamus. This
pattern possibly differed from the hypometabolism pattern of FDG. [(11)C]MPDX
PET is valuable for the detection of degeneration in the temporal and medial
temporal cortices and corticothalamic transmission, and may provide a different
diagnostic tool from FDG-PET in brain disorders such as Alzheimer's disease. Beta-amyloid (Abeta) imaging has great potential to aid in the diagnosis of
Alzheimer disease and the development of therapeutics. The radiation dosimetry
of Abeta radioligands may influence their application; therefore, we calculated
and compared the effective doses (EDs) of 11C-PiB and a new 18F-labeled ligand,
18F-BAY94-9172.
METHODS: Attenuation-corrected whole-body scans were performed at 0, 15, 30, 45,
and 60 min after injection of 350+/-28 MBq (mean+/-SD) of 11C-PiB in 6 subjects
and at 0, 20, 60, 120, and 180 min after injection of 319+/-27 MBq of
18F-BAY94-9172 in 3 subjects. Coregistered CT was used to define volumes of
interest (VOIs) on the PET images. The source organs were the brain, lungs,
liver, kidneys, spleen, and vertebrae. The VOIs for the contents of the
gallbladder, urinary bladder, lower large intestine, upper large intestine, and
small intestine were also defined. Total activity in each organ at each time
point was calculated by use of reference organ volumes. The resultant
time-activity curves were fitted with constrained exponential fits, and
cumulated activities were determined. A dynamic bladder voiding model was used.
The OLINDA/EXM program was used to calculate the whole-body EDs from the
acquired data.
RESULTS: For 11C-PiB, the highest absorbed doses were in the gallbladder wall
(44.80+/-29.30 microGy/MBq), urinary bladder wall (26.30+/-8.50 microGy/MBq),
liver (19.88+/-3.58 microGy/MBq), and kidneys (12.92+/-3.37 microGy/MBq). The ED
was 5.29+/-0.66 microSv/MBq. For 18F-BAY94-9172, the highest doses were also in
the gallbladder wall (132.40+/-43.40 microGy/MBq), urinary bladder wall
(24.77+/-7.36 microGy/MBq), and liver (39.07+/-8.31 microGy/MBq). The ED was
14.67+/-1.39 microSv/MBq.
CONCLUSION: The estimated organ doses for 11C-PiB were comparable to those
reported in earlier research. With the doses used in published studies (300-700
MBq), the EDs would range from 1.6 to 3.7 mSv. The ED of 18F-BAY94-9172 was 30%
lower than that of 18F-FDG and, at the published dose of 300 MBq, would yield an
ED of 4.4 mSv. The dosimetry of both Abeta radioligands is suitable for clinical
and research applications. Traditionally, immunohistochemistry, immunoblotting, and histoblot have been
used to detect protein in tissue samples. However, each of these techniques has
a number of disadvantages. The sensitivity of protein detection in
immunohistochemistry is lost due to fixation or paraffin embedding methods that
modify antigenic sites. The anatomical resolution and specific cellular
involvement are lost in immunoblotting. Histoblot, a hybrid of these two
techniques, is able to resolve these issues, but it cannot be applied to
formalin-fixed tissues. A recent technique, paraffin-embedded tissue (PET) blot,
retains the superior protein detection and anatomical resolution of histoblot
and is applicable to formalin-fixed tissues. Unfortunately, a major obstacle to
the widespread application of PET is the lack of a detailed methodological
description. In this paper, we describe a PET blotting method that was
formulated from our own empirical and experimental research in Alzheimer disease
and a systematic review of the current literature. From this, we conclude that
PET can be applied to a variety of conditions with a wide spectrum of pathology. BACKGROUND: The hypothesis that amyloid deposition is the leading cause of
Alzheimer's disease (AD) is supported by findings in transgenic animal models
and forms the basis of clinical trials of anti-amyloid agents. According to this
theory, amyloid deposition causes severe damage to neurons many years before
onset of dementia via a cascade of several downstream effects. This hypothesis
has, however, not yet been directly tested in human beings because of the very
limited possibility of diagnosing amyloid deposition in vivo, which until
recently required either brain biopsy or PET imaging with an on-site cyclotron
and radiochemistry laboratory. Moreover, a clinical diagnosis of AD requires
that patients have dementia, at which stage any effective treatment aimed at
reducing amyloid deposition will probably be too late.
RECENT DEVELOPMENTS: The amyloid imaging tracers flutemetamol, florbetapir, and
florbetaben labelled with (18)F have been developed for PET; they can be
produced commercially at central cyclotron sites and subsequently delivered to
clinical PET scanning facilities. These tracers are currently undergoing formal
clinical trials to establish whether they can be used to accurately image
fibrillary amyloid and to distinguish patients with AD from normal controls and
those with other diseases that cause dementia. They might also be used as
biomarkers to predict development of AD before onset of dementia and to assess
the effect of anti-amyloid therapy. Negative amyloid scans indicate absence of
AD with a high level of accuracy, but healthy elderly volunteers might have
positive amyloid scans, so their predictive value in isolation is less clear.
Close association of in-vivo amyloid imaging results with post-mortem
histopathological findings was shown with florbetapir in a phase 3 study. WHERE
NEXT?: Therapeutic studies of anti-amyloid agents that include amyloid tracers
as biomarkers are expected to be useful for drug development and to clarify the
relation between amyloid removal and clinical effects. Once the (18)F tracers
become available for diagnostic use, large-scale longitudinal studies will be
needed to clarify their prognostic and diagnostic power in relation to age, risk
factors, and AD subtypes. Ultimately, these tracers will hopefully clarify the
pathophysiological role of amyloid in AD and contribute to development of new
treatments. Amyloid imaging with (18)F-labeled radiotracers will allow widespread use,
facilitating research, diagnosis, and therapeutic development for Alzheimer
disease. The purpose of the study program was to compare cortical amyloid
deposition using (18)F-florbetaben and PET in controls and subjects with mild
cognitive impairment (MCI), frontotemporal lobar degeneration (FTLD), dementia
with Lewy bodies (DLB), vascular dementia (VaD), Parkinson disease (PD), and
Alzheimer disease (AD).
METHODS: One hundred nine subjects in 3 clinical studies at Austin Health were
reviewed: 32 controls, 20 subjects with MCI, and 30 patients with AD, 11 with
FTLD, 7 with DLB, 5 with PD, and 4 with VaD underwent PET after intravenous
injection of 300 MBq of (18)F-florbetaben. Standardized uptake value ratios
(SUVR) using the cerebellar cortex as a reference region were calculated between
90 and 110 min after injection.
RESULTS: When compared with the other groups, AD patients demonstrated
significantly higher SUVRs (P < 0.0001) in neocortical areas. Most AD patients
(96%) and 60% of MCI subjects showed diffuse cortical (18)F-florbetaben
retention. In contrast, only 9% of FTLD, 25% of VaD, 29% of DLB, and no PD
patients and 16% of controls showed cortical binding. Although there was a
correlation between Mini Mental State Examination and β-amyloid burden in the
MCI group, no correlation was observed in controls, FTLD or AD.
CONCLUSION: (18)F-florbetaben had high sensitivity for AD, clearly distinguished
patients with FTLD from AD, and provided results comparable to those reported
with (11)C-Pittsburgh Compound B in a variety of neurodegenerative diseases. (18)F-flutemetamol is a positron emission tomography (PET) tracer for in vivo
amyloid imaging. The ability to classify amyloid scans in a binary manner as
'normal' versus 'Alzheimer-like', is of high clinical relevance. We evaluated
whether a supervised machine learning technique, support vector machines (SVM),
can replicate the assignments made by visual readers blind to the clinical
diagnosis, which image components have highest diagnostic value according to SVM
and how (18)F-flutemetamol-based classification using SVM relates to structural
MRI-based classification using SVM within the same subjects. By means of SVM
with a linear kernel, we analyzed (18)F-flutemetamol scans and volumetric MRI
scans from 72 cases from the (18)F-flutemetamol phase 2 study (27 clinically
probable Alzheimer's disease (AD), 20 amnestic mild cognitive impairment (MCI),
25 controls). In a leave-one-out approach, we trained the (18)F-flutemetamol
based classifier by means of the visual reads and tested whether the classifier
was able to reproduce the assignment based on visual reads and which voxels had
the highest feature weights. The (18)F-flutemetamol based classifier was able to
replicate the assignments obtained by visual reads with 100% accuracy. The
voxels with highest feature weights were in the striatum, precuneus, cingulate
and middle frontal gyrus. Second, to determine concordance between the gray
matter volume- and the (18)F-flutemetamol-based classification, we trained the
classifier with the clinical diagnosis as gold standard. Overall sensitivity of
the (18)F-flutemetamol- and the gray matter volume-based classifiers were
identical (85.2%), albeit with discordant classification in three cases.
Specificity of the (18)F-flutemetamol based classifier was 92% compared to 68%
for MRI. In the MCI group, the (18)F-flutemetamol based classifier distinguished
more reliably between converters and non-converters than the gray matter-based
classifier. The visual read-based binary classification of (18)F-flutemetamol
scans can be replicated using SVM. In this sample the specificity of
(18)F-flutemetamol based SVM for distinguishing AD from controls is higher than
that of gray matter volume-based SVM. The number of elderly patients seeking clinical treatment for memory problems
will rise sharply in coming years as our population ages. These patients present
a challenge for diagnosis and prognosis since cognitive problems in older
patients can arise from many etiologies, some of which are curable. With the
development of clinically available biomarkers for detecting Alzheimer's disease
pathology in living patients, evaluation of cognitively impaired elderly
patients is about to undergo a major paradigm shift. This article describes the
two classes of biomarkers available for assessing Alzheimer's disease risk:
those that indicate presence of amyloid pathology and those that provide
evidence of neuronal injury and neurodegeneration. We argue that, currently,
incorporation of biomarkers of neurodegeneration can help in patient prognosis
whereas tests for amyloid, if used in isolation, have potential for harm.
Amyloid tests are clinically useful only when evidence suggests progressive
cognitive decline or neurodegeneration. Recent trends in neurodegeneration research have been aimed at developing new
amyloid ligands for the neuroimaging of dementia. Among the positron emission
tomography (PET) radiotracers, fluorodeoxyglucose F 18 ((18)F-FDG) is the
compound most widely used in the diagnosis of neurodegenerative dementias.
However, this compound shows a level of specificity and sensitivity for early
Alzheimer's disease detection that is lower than that provided by high-affinity
ligands for β-amyloid (Aβ). Among the new widely available fluorine 18
((18)F)-labeled Aβ ligands, florbetapir F 18 ((18))F-AV-45; Amyvid™) showed
clear qualitative and quantitative correlations between in vivo PET imaging and
postmortem histopathologic analysis of Aβ. Florbetapir F 18 stands out for its
high Aβ affinity and its pharmacokinetic properties that allow 10-minute PET
scan imaging within 90 minutes after administration (dose = 370 MBq).
Importantly, no safety concerns for florbetapir F 18 were found in preclinical
studies. In 2012, the U.S. Food and Drug Administration (FDA) approved Amyvid as
a radiotracer helpful for excluding the presence of Aβ in the brain. It was then
approved earlier this year by the European Medicines Agency (EMA). OBJECTIVE: We examined agreement and disagreement between 2 biomarkers of
β-amyloid (Aβ) deposition (amyloid positron emission tomography [PET] and
cerebrospinal fluid [CSF] Aβ1-42 ) in normal aging and dementia in a large
multicenter study.
METHODS: Concurrently acquired florbetapir PET and CSF Aβ were measured in
cognitively normal, mild cognitive impairment (MCI), and Alzheimer's disease
participants (n = 374) from the Alzheimer's Disease Neuroimaging Initiative. We
also compared Aβ measurements in a separate group with serial CSF measurements
over 3.1 ± 0.8 years that preceded a single florbetapir session. Additional
biomarker and cognitive data allowed us to further examine profiles of
discordant cases.
RESULTS: Florbetapir and CSF Aβ were inversely correlated across all diagnostic
groups, and dichotomous measurements were in agreement in 86% of subjects. Among
subjects showing the most disagreement, the 2 discordant groups had different
profiles: the florbetapir(+) /CSF Aβ(-) group was larger (n = 13) and was made
up of only normal and early MCI subjects, whereas the florbetapir(-) /CSF Aβ(+)
group was smaller (n = 7) and had poorer cognitive function and higher CSF tau,
but no ApoE4 carriers. In the longitudinal sample, we observed both stable
longitudinal CSF Aβ trajectories and those actively transitioning from normal to
abnormal, but the final CSF Aβ measurements were in good agreement with
florbetapir cortical retention.
INTERPRETATION: CSF and amyloid PET measurements of Aβ were consistent in the
majority of subjects in the cross-sectional and longitudinal populations. Based
on our analysis of discordant subjects, the available evidence did not show that
CSF Aβ regularly becomes abnormal prior to fibrillar Aβ accumulation early in
the course of disease. Frontotemporal lobar degeneration (FTLD) represents a group of clinically,
neuropathologically and genetically heterogeneous disorders with plenty of
overlaps between the neurodegenerative mechanism and the clinical phenotype.
FTLD is pathologically characterized by the frontal and temporal lobar atrophy.
Frontotemporal dementia (FTD) clinically presents with abnormalities of behavior
and personality and language impairments variants. The clinical spectrum of FTD
encompasses distinct canonical syndromes: behavioural variant of FTD (bvFTD) and
primary progressive aphasia. The later includes nonfluent/agrammatic variant PPA
(nfvPPA or PNFA), semantic variant PPA (svPPA or SD) and logopenic variant PPA
(lvPPA). In addition, there is also overlap of FTD with motor neuron disease
(FTD-MND or FTD-ALS), as well as the parkinsonian syndromes, progressive
supranuclear palsy (PSP) and corticobasal syndrome (CBS). The FTLD spectrum
disorders are based upon the predomit neuropathological proteins (containing
inclusions of hyperphosphorylated tau or ubiquitin protein, e.g transactive
response (TAR) DNA-binding protein 43 kDa (TDP-43) and fusedin-sarcoma protein
in neurons and glial cells) into three main categories: (1)
microtubule-associated protein tau (FTLD-Tau); (2) TAR DNA-binding protein-43
(FTLD-TDP); and (3) fused in sarcoma protein (FTLD-FUS). There are five main
genes mutations leading clinical and pathological variants in FTLD that
identified by molecular genetic studies, which are chromosome 9 open reading
frame 72 (C9ORF72) gene, granulin (GRN) gene, microtubule associated protein tau
gene (MAPT), the gene encoding valosin-containing protein (VCP) and the charged
multivesicular body protein 2B (CHMP2B). In this review, recent advances on the
different clinic variants, neuroimaging, genetics, pathological subtypes and
clinicopathological associations of FTD will be discussed. BACKGROUND: Although it is well known that many clinical and genetic factors
have been associated with beta-amyloid deposition, few studies have examined the
interactions of such factors across different stages of Alzheimer's
pathogenesis.
METHODS: We used 18F-florbetapir F18 PET imaging to quantify neuritic
beta-amyloid plaque density across four cortical regions in 602 elderly (55-94
years) subjects from the national ADNI biomarker study. The group comprised of
194 normal elderly, 212 early mild cognitive impairment [EMCI], 132 late mild
cognitive impairment [LMCI], and 64 mild Alzheimer's (AD).
FINDINGS: In a model incorporating multiple predictive factors, the effect of
apolipoprotein E ε4 and diagnosis was significant on all four cortical regions.
The highest signals were seen in cingulate followed by frontal and parietal with
lowest signals in temporal lobe (p<0.0001). The effect of apolipoprotein E ε4
(Cohen's D 0.96) on beta-amyloid plaque density was approximately twice as large
as the effect of a diagnosis of AD (Cohen's D 0.51) and thrice as large as the
effect of a diagnosis of LMCI (Cohen's D 0.34) (p<0.0001). Surprisingly, ApoE
ε4+ normal controls had greater mean plaque density across all cortical regions
than ε4- EMCI and ε4- LMCI (p<0.0001, p=0.0009) and showed higher, though
non-significant, mean value than ε4- AD patients (p<0.27). ApoE ε4+ EMCI and
LMCI subjects had significantly greater mean plaque density across all cortical
regions than ε4- AD patients (p<0.027, p<0.0001).
INTERPRETATION: Neuritic amyloid plaque load across progressive clinical stages
of AD varies strongly by ApoE4 genotype. These findings support the need for
better pathology-based and supported diagnosis in routine practice. Our data
also provides additional evidence for a temporal offset between amyloid
deposition and clinically relevant symptoms. BACKGROUND: Early onset Alzheimer's type dementia (EOAD) is usually familial and
associated with mutations in the Presenilin-1 (PSEN1), Presenilin-2 (PSEN2) or
amyloid precursor protein (APP) genes. It is rarely reported in patients of
Hispanic descent.
CASE REPORT: A 49-year-old Hispanic male developed significant cognitive
impairment over a 4-year period. PET scan showed diminished metabolic activity
in the posterior parietal/temporal lobes. Genetic testing revealed the presence
of a PSEN1 gene mutation.
CONCLUSION: Disparities in health care may account for an under-recognition of
EOAD in the Hispanic population. Clinicians should test for EOAD in all patients
with appropriate symptomatology, regardless of ethnicity. Early recognition and
enrollment in clinical trials is vital to enhancing our understanding of the
natural history and treatment of this condition. The introduction of florbetapir (Amyvid) positron emission tomography biomarkers
could dramatically change how we clinically evaluate young patients who present
with nonamnestic cognitive complaints for possible Alzheimer disease (AD).
Logopenic progressive aphasia (LPA) may be the most common example of this type
of clinical problem. In most, but not all, cases, LPA is an early-onset AD
variant presenting with language changes that can be difficult to distinguish
from other progressive aphasias. We clinically evaluated 3 patients with LPA, in
comparison with age-matched and severity-matched patients with typical amnestic
AD, using florbetapir amyloid neuroimaging. The fluorodeoxyglucose-positron
emission tomography scans of LPA patients revealed focal hypometabolism in the
left temporoparietal areas, and the florbetapir scans were diffusely positive
for the presence of amyloid deposition. The florbetapir scans did not differ in
distribution between patients with LPA and those with typical amnestic AD.
Clinical amyloid imaging, although lacking localizing value, is a major advance
in the assessment of early-onset and nonamnestic patients for the presence of
β-amyloid Alzheimer pathology. Author information:
(1)From the Departments of Psychiatry and Neurology (W.E.K.), University of
Pittsburgh, PA; and the Department of Nuclear Medicine and Division of
Neuroscience (D.P.), Vita-Salute San Raffaele University, San Raffaele
Scientific Institute, Milan, Italy. IMPORTANCE: Criteria for preclinical Alzheimer disease (AD) propose β-amyloid
(Aβ) plaques to initiate neurodegeneration within AD-affected regions. However,
some cognitively normal older individuals harbor neural injury similar to
patients with AD, without concurrent Aβ burden. Such findings challenge the
proposed sequence and suggest that Aβ-independent precursors underlie AD-typical
neurodegenerative patterns. OBJECTIVE To examine relationships between Aβ and
non-Aβ factors as well as neurodegeneration within AD regions in cognitively
normal older adults. The study quantified neurodegenerative abnormalities using
imaging biomarkers and examined cross-sectional relationships with Aβ
deposition; white matter lesions (WMLs), a marker of cerebrovascular disease;
and cognitive functions.
DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study in a community-based
convenience sample of 72 cognitively normal older individuals (mean [SD] age,
74.9 [5.7] years; 48 women; mean [SD] 17.0 [1.9] years of education) of the
Berkeley Aging Cohort.
INTERVENTION: Each individual underwent a standardized neuropsychological test
session, magnetic resoce imaging, and positron emission tomography scanning.
MAIN OUTCOMES AND MEASURES: For each individual, 3 AD-sensitive
neurodegeneration biomarkers were measured: hippocampal volume, glucose
metabolism, and gray matter thickness, the latter 2 sampled from cortical
AD-affected regions. To quantify neurodegenerative abnormalities, each biomarker
was age adjusted, dichotomized into a normal or abnormal status (using cutoff
thresholds derived from an independent AD sample), and summarized into 0, 1, or
more than 1 abnormal neurodegenerative biomarker. Degree and topographic
patterns of neurodegenerative abnormalities were assessed and their
relationships with cognitive functions, WML volume, and Aβ deposition
(quantified using carbon 11-labeled Pittsburgh compound B positron emission
tomography).
RESULTS: Of our cognitively normal elderly individuals, 40% (n = 29) displayed
at least 1 abnormal neurodegenerative biomarker, 26% (n = 19) of whom had no
evidence of elevated Pittsburgh compound B retention. In those people who were
classified as having abnormal cortical thickness, degree and topographic
specificity of neurodegenerative abnormalities were similar to patients with AD.
Accumulation of neurodegenerative abnormalities was related to poor memory and
executive functions as well as larger WML volumes but not elevated Pittsburgh
compound B retention.
CONCLUSIONS AND RELEVANCE: Our study confirms that a substantial proportion of
cognitively normal older adults harbor neurodegeneration, without Aβ burden.
Associations of neurodegenerative abnormalities with cerebrovascular disease and
cognitive performance indicate that neurodegenerative pathology can emerge
through non-Aβ pathways within regions most affected by AD. Major imaging biomarkers of Alzheimer's disease include amyloid deposition
[imaged with [(11)C]Pittsburgh compound B (PiB) PET], altered glucose metabolism
(imaged with [(18)F]fluro-deoxyglucose PET), and structural atrophy (imaged by
MRI). Recently we published the initial subset of imaging findings for specific
regions in a cohort of individuals with autosomal domit Alzheimer's disease.
We now extend this work to include a larger cohort, whole-brain analyses
integrating all three imaging modalities, and longitudinal data to examine
regional differences in imaging biomarker dynamics. The anatomical distribution
of imaging biomarkers is described in relation to estimated years from symptom
onset. Autosomal domit Alzheimer's disease mutation carrier individuals have
elevated PiB levels in nearly every cortical region 15 y before the estimated
age of onset. Reduced cortical glucose metabolism and cortical thinning in the
medial and lateral parietal lobe appeared 10 and 5 y, respectively, before
estimated age of onset. Importantly, however, a divergent pattern was observed
subcortically. All subcortical gray-matter regions exhibited elevated PiB
uptake, but despite this, only the hippocampus showed reduced glucose
metabolism. Similarly, atrophy was not observed in the caudate and pallidum
despite marked amyloid accumulation. Finally, before hypometabolism, a
hypermetabolic phase was identified for some cortical regions, including the
precuneus and posterior cingulate. Additional analyses of individuals in which
longitudinal data were available suggested that an accelerated appearance of
volumetric declines approximately coincides with the onset of the symptomatic
phase of the disease. Cholesterol is implicated in the development of late-onset Alzheimer's disease
(AD). We sought to determine the associations between beta amyloid (Aβ) plaque
deposition in vivo using Pittsburgh compound B (PiB) and several indices of
cholesterol homeostasis (i.e., total cholesterol, high-density lipoprotein
cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein
E (ApoE), clusterin, oxysterol metabolites of cholesterol, and previously
reported genes associated with late-onset AD) in 175 nondemented elderly
subjects. High Aβ deposition was associated significantly with a lower
Mini-Mental State Examination score (<27 points, p = 0.04), high systolic blood
pressure (p = 0.04), carrying the apolipoprotein E epsilon 4 allele (p < 0.01),
and lower plasma ApoE levels (p = 0.02), and variation in the ABCA7 (p = 0.02)
and EPHA1 genes (p = 0.02). Cholesterol measures were not related to Aβ
deposition in this cohort of nondemented elderly adults. However, plasma and
genetic factors relating to cholesterol transport were associated with Aβ
deposition in the brain. A better understanding of cholesterol transport
mechanisms may lead to the design of potential targets for the prevention of Aβ
deposition in the brain. |
What was the purpose of the FANTOM3 project? | The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species | The international FANTOM consortium aims to produce a comprehensive picture of
the mammalian transcriptome, based upon an extensive cDNA collection and
functional annotation of full-length enriched cDNAs. The previous dataset,
FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation
revealed that this cDNA dataset contained only about half of the estimated
number of mouse protein-coding genes, indicating that a number of cDNAs still
remained to be collected and identified. To pursue the complete gene catalog
that covers all predicted mouse genes, cloning and sequencing of full-length
enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly
isolated cDNAs were subjected to functional annotation, and the annotation of
4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation,
we improved our automated annotation pipeline by introducing new coding sequence
prediction programs and developed a Web-based annotation interface for
simplifying the annotation procedures to reduce manual annotation errors.
Automated coding sequence and function prediction was followed with manual
curation and review by expert curators. A total of 102,801 full-length enriched
mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally
annotated as protein coding (including partial or truncated transcripts),
providing to our knowledge the greatest current coverage of the mouse proteome
by full-length cDNAs. The total number of distinct non-protein-coding
transcripts increased to 34,030. The FANTOM3 annotation system, consisting of
automated computational prediction, manual curation, and final expert curation,
facilitated the comprehensive characterization of the mouse transcriptome, and
could be applied to the transcriptomes of other species. |
In which isochores are Alu elements enriched? | Alu elements are enriched in high GC% isochores due to reduced Alu loss by recombination in these regions. The frequency of Alu sequences increases with increasing GC, but attains a maximum in H2 isochores. | We investigated the genomic distribution of mouse and human repeated sequences
by assessing their relative amounts in the four major components into which
these genomes can be resolved by density gradient centrifugation techniques.
These components are families of fragments that account for most or all of
main-band DNAs, range in dG + dC content from 37% to 49%, and are derived by
preparative breakage from long DNA segments (greater than 300 kb) of fairly
homogeneous composition, the isochores. The results indicate that the short
repeats of the B1 family of mouse and of the Alu I family of man are most
frequent in the heavy components, whereas the long repeats of the BamHI family
of mouse and of the Kpn I family of man are mainly present in the two light
components. These results show that the genomic distribution of repeated
sequences is nonuniform and conserved in two mammalian species. In addition, we
observed that the base composition of two classes of repeats (60% dG + dC for
short repeats; 39% dG + dC for long repeats) is correlated with the composition
of the major components in which they are embedded. Finally, we obtained
evidence that not only the short repeats but also the long repeats are
transcribed, these transcripts having been found in mouse poly(A)+ mRNA. As part of an effort to identify the gene responsible for the predomit form
of polycystic kidney disease (PKD1), we used a gridded human P1 library for
contig assembly. The interval of interest, a 700-kb segment on chromosome
16p13.3, can be physically delineated by the genetic markers D16S125 and D16S84
and chromosomally characterized as a GC-rich isochore enriched for CpG islands,
genes, and Alu-like repeats. Our attempts to recover CEPH YACs that encode this
region of chromosome 16 were unsuccessful. However, we screened an arrayed P1
library using 15 distinct probes from the D16S125-D16S84 interval and identified
56 independent P1 clones. Only one probe from the interval was unsuccessful in
identifying a P1 clone. Forty-four P1 clones were determined to be unique based
on restriction enzyme analysis, and 42 of these were found to originate from
chromosome 16p13.3, based on FISH to metaphase chromosomes. The 700-kb interval
could be defined by a single sequence-ready contig comprised of 12 P1 clones and
1 cosmid clone. Our studies support the use of multiple libraries to generate
the requisite physical reagents for positional cloning and encourage the use of
Escherichia coli-based large-insert cloning systems to recover clones from
YAC-deficient chromosomal intervals. Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly
distributed in the human genome. The majority of GC-poor LINEs reside in the
GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores.
The discovery that LINES and Alus share similar target site duplication and a
common AT-rich insertion site specificity raised the question as to why these
two families of repeats show such a different distribution in the genome. This
problem was investigated here by studying the isochore distributions of
subfamilies of LINES and Alus characterized by different degrees of divergence
from the consensus sequences, and of Alus, LINEs and pseudogenes located on
chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the
genome than old Alus. This suggests that the gradual accumulation of Alus in
GC-rich isochores has occurred because of their higher stability in
compositionally matching chromosomal regions. Densities of Alus and LINEs
increase and decrease, respectively, with increasing GC levels, except for the
telomeric regions of the analyzed chromosomes. In addition to LINEs, processed
pseudogenes are also more frequent in GC-poor isochores. Finally, the present
results on Alu and LINE stability/exclusion predict significant losses of Alu
DNA from the GC-poor isochores during evolution, a phenomenon apparently due to
negative selection against sequences that differ from the isochore composition. Alu retrotransposons do not show a homogeneous distribution over the human
genome but have a higher density in GC-rich (H) than in AT-rich (L) isochores.
However, since they preferentially insert into the L isochores, the question
arises: What is the evolutionary mechanism that shifts the Alu density maximum
from L to H isochores? To disclose the role played by each of the potential
mechanisms involved in such biased distribution, we carried out a genome-wide
analysis of the density of the Alus as a function of their evolutionary age,
isochore membership, and intron vs. intergene location. Since Alus depend on the
retrotransposase encoded by the LINE1 elements, we also studied the distribution
of LINE1 to provide a complete evolutionary scenario. We consecutively check,
and discard, the contributions of the Alu/LINE1 competition for
retrotransposase, compositional matching pressure, and Alu overrepresentation in
introns. In analyzing the role played by unequal recombination, we scan the
genome for Alu trimers, a direct product of Alu-Alu recombination. Through
computer simulations, we show that such trimers are much more frequent than
expected, the observed/expected ratio being higher in L than in H isochores.
This result, together with the known higher selective disadvantage of
recombination products in H isochores, points to Alu-Alu recombination as the
main agent provoking the density shift of Alus toward the GC-rich parts of the
genome. Two independent pieces of evidence-the lower evolutionary divergence
shown by recently inserted Alu subfamilies and the higher frequency of old
stand-alone Alus in L isochores-support such a conclusion. Other evolutionary
factors, such as population bottlenecks during primate speciation, may have
accelerated the fast accumulation of Alus in GC-rich isochores. BACKGROUND: The very recent availability of fully sequenced individual human
genomes is a major revolution in biology which is certainly going to provide new
insights into genetic diseases and genomic rearrangements.
RESULTS: We mapped the insertions, deletions and SNPs (single nucleotide
polymorphisms) that are present in Craig Venter's genome, more precisely on
chromosomes 17 to 22, and compared them with the human reference genome hg17.
Our results show that insertions and deletions are almost absent in L1 and
generally scarce in L2 isochore families (GC-poor L1+L2 isochores represent
slightly over half of the human genome), whereas they increase in GC-rich
isochores, largely paralleling the densities of genes, retroviral integrations
and Alu sequences. The distributions of insertions/deletions are in striking
contrast with those of SNPs which exhibit almost the same density across all
isochore families with, however, a trend for lower concentrations in gene-rich
regions.
CONCLUSIONS: Our study strongly suggests that the distribution of
insertions/deletions is due to the structure of chromatin which is mostly open
in gene-rich, GC-rich isochores, and largely closed in gene-poor, GC-poor
isochores. The different distributions of insertions/deletions and SNPs are
clearly related to the two different responsible mechanisms, namely
recombination and point mutations. The distribution in the human genome of the largest family of mobile elements,
the Alu sequences, has been investigated for the past 30 years, and the vast
majority of Alu sequences were shown to have the highest density in GC-rich
isochores. Ten years ago, it was discovered, however, that the small "youngest"
(most recently transposed) Alu families had a strikingly different distribution
compared with the "old" families. This raised the question as to how this change
took place in evolution. We solved what was considered to be a "mystery" by 1)
revisiting our previous results on the integration and stability of retroviral
sequences, and 2) assessing the densities of acceptor sites TTTT/AA in isochore
families. We could conclude 1) that the open state of chromatin structure plays
a crucial role in allowing not only the initial integration of retroviral
sequences but also that of the youngest Alu sequences, and 2) that the
distribution of old Alus can be explained as due to Alu sequences being unstable
in the GC-poor isochores but stable in the compositionally matching GC-rich
isochores, again in line with what happens in the case of retroviral sequences. |
Is oxalate renal excretion increased after bariatric surgery? | Bariatric surgery is associated with a significant risk of nephrolithiasis.
Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy must be considered with the other risks of bariatric surgery
Hyperoxaluria in patients treated with bariatric surgery was found to be a result of hyperabsorption of oxalate | Hyperoxaluria and hypercalciuria are common features of renal calcium stone
disease. The purpose of the present investigation was to examine the
relationships between the intestinal absorption and the renal handling of
oxalate and calcium in patients with idiopathic renal stone disease and in
patients with enteric hyperoxaluria following jejunoileal bypass (JIB), in
comparison with healthy controls. Hyperoxaluria was associated with a higher
frequency of both stone episodes and stone operations than a lower urinary
oxalate concentration. Patients with idiopathic stone disease showed increased
intestinal uptake of both oxalate and calcium, which was probably of importance
for their propensity to form calcium oxalate-containing stones. Hyperoxaluria in
patients with JIB was found to be a result of hyperabsorption of oxalate, and
these patients displayed altered oxalate kinetics with continued urinary
excretion of orally administered 14C-oxalate for more than 48 hours. The
prolonged excretion is assumed to be due to a prolonged absorption and/or an
increased oxalate pool. Malabsorption of calcium and low fasting urinary calcium
excretion in the JIB patients were associated with high tubular reabsorption of
calcium, the latter presumably attributable to a compensatory increase in
circulating parathyroid hormone (PTH). In most recurrent renal stone formers the
urinary calcium concentration was increased, with an inverse relationship to
serum PTH, indicating intestinal hyperabsorption of calcium. A subgroup of
hypercalciuric patients showed increased urinary calcium due to reduced tubular
reabsorption of calcium. It is suggested that this is a renal defect resulting
in a compensatory rise in PTH. Two different mechanisms of similar prevalence
might explain enhanced secretion of PTH in normocalcaemic stone disease, namely
reduced calcium absorption and a renal defect in the form of reduced tubular
reabsorption of calcium. Glycosaminoglycans efficiently inhibit calcium oxalate
crystal growth by binding to the surface of calcium oxalate crystals. In this
study the binding was dependent on ionic strength. Higher affinity to the
crystals may be the reason why highly charged glycosaminoglycans were more
efficient inhibitors of calcium oxalate crystal growth. A calcium-containing
organic marine hydrocolloid with the capacity to bind oxalate in vitro was shown
to reduce enteric hyperoxaluria. In addition to biochemical effects considerable
improvements in diarrhoeal symptoms were reported. BACKGROUND: Neither the presence nor prevalence of enteric hyperoxaluria has
been recognized after Roux-en-Y gastric bypass (RYGBP). We have noted a high
rate of oxalate nephrolithiasis and even 2 patients with oxalate nephropathy in
this patient population postoperatively. Our aim was to determine the frequency
of the occurrence and effects of enteric hyperoxaluria after RYGBP.
METHODS: Retrospective review of all patients at our institution diagnosed with
calcium oxalate nephrolithiasis or oxalate nephropathy after standard (n = 14)
or distal (n = 9) RYGBP. The mean postoperative follow-up was 55 months.
RESULTS: A total of 23 patients (14 men and 9 women; mean age 45 years; mean
preoperative body mass index 55 kg/m(2)) developed enteric hyperoxaluria after
RYGBP, defined by the presence of oxalate nephropathy (n = 2) or calcium oxalate
nephrolithiasis (n = 21) and increased 24-hour excretion of urinary oxalate
and/or calcium oxalate supersaturation. Enteric hyperoxaluria was recognized
after a mean weight loss of 46 kg at 29 months (range 2-85) after RYGBP. Two
patients developed renal failure and required chronic hemodialysis. Of the 21
patients with nephrolithiasis, 14 had no history of nephrolithiasis
preoperatively, and 19 of 21 required lithotripsy or other intervention. Of the
23 patients, 20 tested had increased oxalate excretion, and 14 of 15 tested had
high urine calcium oxalate supersaturation.
CONCLUSION: Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy must
be considered with the other risks of RYGBP. Efforts should be made to identify
factors that predispose patients to developing hyperoxaluria. PURPOSE: Nephrolithiasis and renal failure secondary to severe hyperoxaluria
were complications of jejunoileal bypass for obesity, leading to the
discontinuation of this procedure in the United States in 1980. Bariatric
procedures currently in use have not been adequately evaluated for this
complication.
MATERIALS AND METHODS: We compared 24-hour urine chemistry studies of 132
patients with nephrolithiasis who had undergone bariatric surgery with the urine
chemistry studies of patients who had undergone jejunoileal bypass, those with
routine kidney stones and normal subjects. The primary aim was to determine if
hyperoxaluria developed in patients who underwent bariatric surgery and had
kidney stones as had been seen with jejunoileal bypass.
RESULTS: Patients who have undergone modern bariatric surgery had an adjusted
mean urine oxalate excretion of 83 mg per day compared to 39 mg per day for
routine kidney stone formers and 34 mg per day for normal subjects (p <0.001 for
both comparisons), but not quite as high as that found in patients treated with
jejunoileal bypass (102 mg per day, p <0.001). Urine supersaturation of calcium
oxalate, the main driving force for calcium oxalate stone formation, was higher
in patients treated with bariatric surgery compared to routine kidney stone
formers and normal subjects (p <0.001 for both comparisons).
CONCLUSIONS: Hyperoxaluria is the most significant abnormality of urine
chemistry studies in patients with kidney stones who have undergone bariatric
surgery. Many of these patients have a degree of hyperoxaluria that could lead
to kidney failure. Further studies are required to determine the prevalence of
this problem in patients who have undergone bariatric surgery. BACKGROUND: Patients treated for obesity with jejunoileal bypass (JIB)
experienced a marked increased risk of hyperoxaluria, nephrolithiasis, and
oxalate nephropathy developing. Jejunoileal bypass has been abandoned and
replaced with other options, including Roux-en-Y gastric bypass (RYGB). Changes
in urinary lithogenic risk factors after RYGB are currently unknown. Our purpose
was to determine whether RYGB is associated with elevated risk of developing
calcium oxalate stone formation through increased urinary oxalate excretion and
relative supersaturation of calcium oxalate.
STUDY DESIGN: A prospective longitudinal cohort study of 24 morbidly obese
adults (9 men and 15 women) recruited from a university-based bariatric surgery
clinic scheduled to undergo RYGB between December 2005 and April 2007. Patients
provided 24-hour urine collections for analysis 7 days before and 90 days after
operation. Primary outcomes were changes in 24-hour urinary oxalate excretion
and relative supersaturation of calcium oxalate from baseline to 3 months
post-RYGB.
RESULTS: Compared with their baseline, patients undergoing RYGB had increased
urinary oxalate excretion (31 +/- 10 mg/d versus 41 +/- 18 mg/d; p = 0.026) and
relative supersaturation of calcium oxalate (1.73 +/- 0.81 versus 3.47 +/- 2.59;
p = 0.030) 3 months post-RYGB in six patients (25%). De novo hyperoxaluria
developed. There were no preoperative patient characteristics predictive of
development of de novo hyperoxaluria or the magnitude of change of daily oxalate
excretion.
CONCLUSIONS: This prospective study indicates that RYGB is associated with an
earlier increase in urinary oxalate excretion and relative supersaturation of
calcium oxalate than previously reported. Additional studies are needed to
determine longterm post-RYGB changes in urinary oxalate excretion and identify
patients that might be at risk for hyperoxaluria developing. BACKGROUND AND OBJECTIVES: The most common bariatric surgery is Roux-en-Y
gastric bypass (RYGB), which has been associated with hyperoxaluria and
nephrolithiasis. We report a novel association of RYGB with renal insufficiency
as a result of oxalate nephropathy.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Eleven cases of oxalate
nephropathy after RYGB were identified from the Renal Pathology Laboratory of
Columbia University. The clinical features, pathologic findings, and outcomes
are described.
RESULTS: Patients were predomitly white (72.7%) with a mean age of 61.3 yr.
Indications for RYGB included morbid obesity (eight patients) and reconstruction
after total gastrectomy for gastric cancer (three patients). All 11 patients had
a history of hypertension, and 9 were diabetic. Patients presented with acute
renal failure, often superimposed on mild chronic renal insufficiency (n = 7),
at a median of 12 mo after RYGB. The mean creatinine at baseline, at discovery
of acute renal failure, and at biopsy was 1.5, 5.0, and 6.5 mg/dl, respectively.
Renal biopsies revealed diffuse tubular degenerative changes, abundant tubular
calcium oxalate deposits, and varying degrees of tubulointerstitial scarring. In
addition, seven biopsies had underlying diabetic glomerulosclerosis and two had
glomerulosclerosis attributable to obesity and hypertension. Eight of 11
patients rapidly progressed to ESRD and required hemodialysis at a mean of 3.2
wk after renal biopsy. The remaining three patients were left with significant
chronic kidney disease.
CONCLUSIONS: Oxalate nephropathy is an underrecognized complication of RYGB and
typically results in rapid progression to ESRD. Patients with pre-existing renal
disease may be at higher risk for this complication. PURPOSE: Recent investigations have shown increased oxalate excretion in
patients in whom kidney stones formed after contemporary bariatric surgery. We
determined whether there is an increased prevalence of hyperoxaluria after such
procedures performed in nonstone formers.
MATERIALS AND METHODS: A total of 58 nonstone forming adults who underwent
laparoscopic Roux-en-Y (52) or a biliopancreatic diversion-duodenal switch
procedure (6) collected 24-hour urine specimens 6 months or greater after
bariatric surgery. Standard stone risk parameters were assessed. Comparisons
were made with a group of healthy nonstone forming adults and stone formers in a
commercial database.
RESULTS: The bariatric group had a significantly higher mean urinary oxalate
excretion compared to that in controls and stone formers (67.2 vs 34.1 and 37.0
mg per day, respectively, p <0.001). Mean oxalate excretion of patients who
underwent a biliopancreatic diversion-duodenal switch procedure was higher than
in the Roux-en-Y group (90 vs 62 mg per day, p <0.05). There was a significant
correlation between urine oxalate excretion on the 2 collection days but some
patients showed significant variability. Of the patients 74% showed
hyperoxaluria in at least 1, 24-hour urine collection and 26% demonstrated
profound hyperoxaluria, defined as oxalate excretion more than 100 mg per day,
in at least 1 collection. This occurred in 3 of the 6 patients in the
biliopancreatic diversion-duodenal switch group and in 12 of the 52 in the
Roux-en-Y cohort. Hyperoxaluria was not uniformly expressed.
CONCLUSIONS: There is a high prevalence of hyperoxaluria in patients without a
history of kidney stones who undergo bariatric surgery. A significant proportion
of these patients have profound hyperoxaluria, which is not uniformly expressed. BACKGROUND: Hyperoxaluria and increased calcium oxalate stone formation occur
after Roux-en-Y gastric bypass (RYGB) surgery for morbid obesity. The etiology
of this hyperoxaluria is unknown. We hypothesized that after bariatric surgery,
intestinal hyperabsorption of oxalate contributes to increases in plasma oxalate
and urinary calcium oxalate supersaturation.
METHODS: We prospectively examined oxalate metabolism in 11 morbidly obese
subjects before and 6 and 12 months after RYGB (n = 9) and biliopancreatic
diversion-duodenal switch (BPD-DS) (n = 2). We measured 24-hour urinary
supersaturations for calcium oxalate, apatite, brushite, uric acid, and sodium
urate; fasting plasma oxalate; 72-hour fecal fat; and increases in urine oxalate
following an oral oxalate load.
RESULTS: Six and 12 months after RYGB, plasma oxalate and urine calcium oxalate
supersaturation increased significantly compared with similar measurements
obtained before surgery (all P ≤ .02). Fecal fat excretion at 6 and 12 months
was increased (P = .026 and .055, 0 vs 6 and 12 months). An increase in urine
oxalate excretion after an oral dose of oxalate was observed at 6 and 12 months
(all P ≤ .02). Therefore, after bariatric surgery, increases in fecal fat
excretion, urinary oxalate excretion after an oral oxalate load, plasma oxalate,
and urinary calcium oxalate supersaturation values were observed.
CONCLUSION: Enteric hyperoxaluria is often present in patients after the
operations of RYGB and BPD-DS that utilize an element of intestinal
malabsorption as a mechanism for weight loss. OBJECTIVE: To identify the effect of a controlled metabolic diet on reducing
urinary calcium oxalate (CaOx) supersaturation in subjects with hyperoxaluric
nephrolithiasis after potentially malabsorptive forms of bariatric surgery.
METHODS: Subjects with a history of CaOx kidney stones and mild hyperoxaluria
after bariatric surgery (n = 9) collected baseline 24-hour urine samples while
consuming a free choice diet. They were then instructed to consume a controlled
diet low in oxalate (70-80 mg/d), normal in calcium (1000 mg/d), and moderate in
protein before 2 final 24-hour urine collections.
RESULTS: Overall, the urinary CaOx supersaturation decreased from 1.97 ± 0.49
delta Gibbs (DG) with the free choice diet to 1.13 ± 0.75 DG with the controlled
diet (P < .01). This occurred in the absence of a significant change in urinary
oxalate excretion (0.69 ± 0.29 mmol/d with the free choice diet compared with
0.66 ± 0.38 mmol/d with the controlled diet). Urinary volume, citrate, and pH
all increased, although not significantly (P > .05), contributing to the
significant CaOx supersaturation change.
CONCLUSION: A controlled metabolic diet normal in calcium, moderate in protein,
and reduced in oxalate can positively affect urinary CaOx supersaturation after
bariatric surgery. However, this diet did not appear to decrease urinary oxalate
excretion. Therefore, restriction of dietary oxalate alone might not be enough
to reduce urinary oxalate excretion to normal levels in this group of patients
with known enteric hyperoxaluria. Additional strategies could be necessary, such
as the use of oral calcium supplements as oxalate binders and a lower fat diet. BACKGROUND AND OBJECTIVES: Bariatric surgery (BS) may be associated with
increased oxalate excretion and a higher risk of nephrolithiasis. This study
aimed to investigate urinary abnormalities and responses to an acute oxalate
load as an indirect assessment of the intestinal absorption of oxalate in this
population.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Twenty-four-hour urine specimens
were collected from 61 patients a median of 48 months after BS (post-BS) as well
as from 30 morbidly obese (MO) participants; dietary information was obtained
through 24-hour food recalls. An oral oxalate load test (OLT), consisting of
2-hour urine samples after overnight fasting and 2, 4, and 6 hours after
consuming 375 mg of oxalate (spinach juice), was performed on 21 MO and 22
post-BS patients 12 months after BS. Ten post-BS patients also underwent OLT
before surgery (pre-BS).
RESULTS: There was a higher percentage of low urinary volume (<1.5 L/d) in
post-BS versus MO (P<0.001). Hypocitraturia and hyperoxaluria (P=0.13 and
P=0.36, respectively) were more frequent in BS versus MO patients. The OLT
showed intragroup (P<0.001 for all periods versus baseline) and intergroup
differences (P<0.001 for post-BS versus MO; P=0.03 for post-BS versus pre-BS).
The total mean increment in oxaluria after 6 hours of load, expressed as area
under the curve, was higher in both post-BS versus MO and in post-BS versus
pre-BS participants (P<0.001 for both).
CONCLUSIONS: The mean oxaluric response to an oxalate load is markedly elevated
in post-bariatric surgery patients, suggesting that increased intestinal
absorption of dietary oxalate is a predisposing mechanism for enteric
hyperoxaluria. |
Which are the mutational hotspots of the human KRAS oncogene? | The KRAS oncogene has four main mutational hotspots located at codons 12, 13, 61 and 146. | Medullary thyroid carcinoma (MTC) occurs sporadically (sMTC) or as part of the
inherited cancer syndrome, multiple endocrine neoplasia type 2 (MEN 2). While
the occurence of the MEN 2 syndrome is associated with mutations in the RET
protooncogene, the reason for carcinogenesis in sMTC still remains unclear. Ras
is a frequently mutated oncogene in a broad spectrum of human tumors and has
been found in about 50% of follicular, papillary or anaplastic thyroid
carcinomas. The purpose of this study was to determine, whether mutations in the
ras oncogene could play a possible role in the carcinogenesis of sMTC. In this
study we analyzed 15 sMTC for mutations in the hotspots codon 12, 13 and 61 of
the H- and K-ras oncogene. We used the direct sequencing technique. In none of
the examined tumors we were able to detect a mutation in the codon 12, 13 and 61
of the H-ras and K-ras oncogene. Based upon these results, we conclude that H-
and K-ras do not play an important role in the carcinogenesis of sMTC. Our previous study showed that the mutation hotspots of the K-ras proto-oncogene
in human functional adrenocortical tumors are in codons 15, 16, 18, and 31, thus
differing from the sites in other tumors. In addition, analyzing the K-Ras
protein by a recombit DNA technique showed that the activity of endogenic
GTPase and the GTPase-activating protein (GAP)-binding ability were
significantly decreased in patients with these tumors. The aim of this study was
to understand whether those K-ras mutants, which were found only in human
adrenocortical tumors, play an important role in these tumors. Thus, the mutant
K-ras cDNA was constructed with mammalian expression vectors and transfected
into normal adrenocortical cells. The amount of cortisol secreted by the
transfected cells was 20 to 30 times that of normal cells. Furthermore, Northern
blot analysis revealed that the expression of the three steroidogenesis-related
genes P450(scc) (cholesterol side-chain cleavage enzyme), P450(C17)
(17alpha-hydroxylase/17, 20-lyase), and P450(C21) (steroid 21-hydroxylase) gene
increased in the transfected cells. The K-ras oncogene significantly increases
cortisol secretion by normal adrenocortical cells. Nonsmoking women in Xuan Wei County, Yun Province, China who use smoky coal
for cooking and heating in poorly ventilated homes have the highest lung cancer
mortality rate in China, and their lung cancer is linked epidemiologically to
their use of smoky coal. The emissions contain 81% organic matter, of which 43%
is polycyclic aromatic hydrocarbons (PAHs). Exposure assessment and molecular
analysis of the lung tumors from nonsmoking women who use smoky coal strongly
indicate that PAHs in the emissions are a primary cause of the elevated lung
cancer in this population. Here we have determined the mutation spectra of an
extract of smoky coal emissions in Salmonella TA98 and TA100; the extract was
not mutagenic in TA104. The extract was 8.7 x more mutagenic in TA100 with S9
than without (8.7 rev/microg versus 1.0 rev/microg) and was >3 x more mutagenic
in TA100 than in TA98--consistent with a prominent role for PAHs in the
mutagenicity of the extract because PAHs are generally more mutagenic in the
base-substitution strain TA100 than in the frameshift strain TA98. The extract
induced only a hotspot mutation in TA98; another combustion emission, cigarette
smoke condensate (CSC), also induces this single class of mutation. In TA100,
the mutation spectra of the extract were not significantly different in the
presence or absence of S9 and were primarily (78-86%) GC --> TA transversions.
This mutation is induced to a similar extent by CSC (78%) and the PAH
benzo[a]pyrene (B[a]P) (77%). The frequency of GC --> TA transversions induced
in Salmonella by the extract (78-86%) is similar to the frequency of this
mutation in the TP53 (76%) and KRAS (86%) genes of lung tumors from nonsmoking
women exposed to smoky coal emissions. The mutation spectra of the extract
reflect the presence of PAHs in the mixture and support a role for PAHs in the
induction of the mutations and tumors due to exposure to smoky coal emissions. Aberrant signalling via platelet derived growth factor receptors (PDGFRs) and
the RAS/MAPK pathway has been implicated in the development of medulloblastoma,
the most common maligt brain tumour in childhood. To determine whether
genetic mechanisms play a role in the activation of PDGFR-RAS/MAPK signalling in
medulloblastoma, we performed a direct sequence analysis of the established
mutational "hotspots" of known targets of activating mutations within the
pathway (PDGFRA, NRAS, KRAS, HRAS and BRAF) and PDFRFB, in a cohort of 28
primary tumours. A synonymous sequence variation in PDGFRA (CCG to CCA; PRO 567
PRO) was detected in two cases (approximately 7%), but not in 150 normal
chromosomes assessed, suggesting that the PDGFRA locus may be associated with
medulloblastoma development in certain cases. No evidence for oncogenic
mutations affecting NRAS, KRAS, HRAS, BRAF or PDFRFB was found in any case.
These data demonstrate that activating mutations in established mutational
hotspots within the PDGFR-RAS/MAPK pathway are rare events in medulloblastoma
development, and suggest that alternative mechanisms are responsible for
RAS/MAPK pathway activation in this disease. The mutations C742T, G746T, G747T in the TP53 gene and G35T in the KRAS gene
have been repeatedly found in sectors of human tumors by direct DNA sequencing.
The mutation G508A in the HPRT1 gene has been repeatedly found among peripheral
T lymphocytes by clonal expansion under selective conditions. To discover if
these mutations also occur frequently in normal tissues from which tumors arise,
we have developed and validated allele-specific mismatch amplification mutation
assays (MAMA) for each mutation. Reconstruction experiments demonstrated
linearity in the range of 9-3000 mutant alleles among 3 x 10(6) wild-type
alleles. The cumulative distributions of all negative controls established
robust detection limits (P<0.05) of 34-125 mutants per 10(6) copies assayed
depending on the mutation. One hundred and seventy-seven micro-anatomical
samples of approximately (0.5-6)x10(6) tracheal-bronchial epithelial cells from
nine non-smokers were assayed representing en toto the equivalent of
approximately 1.6 human bronchial trees to the fifth bifurcation. Statistically
significant mutant copy numbers were found in 257 of 463 assays. Clusters of
mutant copies ranged from 10 to 1000 in 239/257 positive samples. As all five
point mutations were detected at mutant fractions of >10(-5) in two or more
lungs, we infer that they are mutational hotspots generated in lung epithelial
stem cells. As the cancer-associated mutations did not differ in cluster size
distribution from the HPRT1 mutation, we infer that none of the mutations
conferred a growth advantage to somatic heterozygous clusters or maintece
turnover units. Specific mutants appeared in very large copy numbers,
1000-35,000, in 18/257 positive assays. Various hypotheses to account for the
observed cluster size distributions are offered. BACKGROUND: Anaplastic thyroid carcinoma (ATC), a rare and highly maligt
tumor, has long been thought to arise from well-differentiated carcinoma (WDC)
such as follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma
(PTC). The purpose of this study was to test this notion by examining whether
and, if so, how often ATC harbors the oncogenes that are commonly associated
with WDC, such as RAS in FTC and BRAF in PTC.
METHODS: We analyzed the mutation hotspots of BRAF (codon 600) and N-, K-, and
H-RAS (codons 12, 13, and 61) in 16 ATCs. We also examined two genes, PIK3CA
(exons 9 and 20) and TP53 (exons 5-9), both of which have been reported in ATCs.
RESULTS: The results showed that approximately 31% (5 of 16) of ATCs harbored
N-RAS mutation, 6% (1 of 16) had mutated BRAF, and approximately 56% (9 of 16)
had mutated TP53. As to the three ATCs that had coexisted PTCs, mutated BRAF was
detected in all PTC components but only in one ATC, while mutated PIK3CA was
found in only one PTC component but not in the ATC.
CONCLUSION: A number of ATCs arise from WDCs, more often from RAS-mutant tumors
than from BRAF-mutant tumors, implying that particular attention should be paid
to the WDC harboring RAS mutation. Activating mutations of the BRAF and KRAS genes cause constitutive stimulation
of an important cell-signaling pathway promoting tumorigenesis, and are
increasingly recognized as determits of response to targeted cancer
therapies. The V600E mutation accounts for most of the BRAF mutations in cancer,
and KRAS mutations are predomitly encoded by nucleotide substitutions within
codons 12 and 13. We designed novel pyrosequencing assays for the detection of
the common "hotspot" mutations in these genes, which demonstrated analytical
sensitivities of <or=10% in titrations of mutant cell lines. The KRAS
pyrosequencing assay has the ability to simultaneously identify all potential
nucleotide changes within the mutation cluster at codons 12 and 13, with a
sequence output in the sense direction to facilitate results interpretation.
These assays were used to determine the mutation status in a prospective series
of 1198 sporadic colorectal cancers. The BRAF V600E mutation was detected in
13.2% of the colorectal cancers. The frequency of KRAS mutations in our cohort
was 32.4%, with G>A transitions at position 2 of codons 12 and 13 being most
prevalent. Both assays proved highly sensitive and specific when applied to
clinical specimens, and were applicable to both fresh-frozen and formalin-fixed
paraffin-embedded archival tissues. These assays would serve as a suitable
platform for large-scale mutation detection in cancer specimens where the
facility for pyrosequencing is available. PURPOSE: The goal of this study was to comprehensively define the incidence of
mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC)
and nonendometrioid endometrial cancer (NEEC).
EXPERIMENTAL DESIGN: We resequenced all coding exons of PIK3CA and PTEN, and
exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations
were confirmed by sequencing matched normal DNAs. The biochemical properties of
a subset of novel PIK3CA mutations were determined by exogenously expressing
wild type and mutant constructs in U2OS cells and measuring levels of
AKT(Ser473) phosphorylation.
RESULTS: Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of
66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons
1-7 and half were in exons 9 and 20. The exons 1-7 mutations localized to the
ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93,
Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93, and
Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E,
-delP449-L455, and -E453K mutants led to increased levels of phospho-AKT(Ser473)
compared to wild-type p110α. Overall, 62% of exons 1-7 PIK3CA mutants and 64% of
exons 9-20 PIK3CA mutants were activating; 72% of exon 1-7 mutations have not
previously been reported in endometrial cancer.
CONCLUSIONS: Our study identified a new subgroup of endometrial cancer patients
with activating mutations in the amino-terminal domains of p110α; these patients
might be appropriate for consideration in clinical trials of targeted therapies
directed against the PI3K pathway. In February 2010, the Medical Advisory Secretariat (MAS) began work on
evidence-based reviews of the literature surrounding three pharmacogenomic
tests. This project came about when Cancer Care Ontario (CCO) asked MAS to
provide evidence-based analyses on the effectiveness and cost-effectiveness of
three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based
analyses have been prepared for each of these technologies. These have been
completed in conjunction with internal and external stakeholders, including a
Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup
committees were developed for each disease area. For each technology, an
economic analysis was also completed by the Toronto Health Economics and
Technology Assessment Collaborative (THETA) and is summarized within the
reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT:
www.health.gov.on.ca/mas or at
www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION
PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST
CANCER: An Evidence-Based and Economic AnalysisEpidermal Growth Factor Receptor
Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine
Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung
Cancer: an Evidence-Based and Economic AnalysisK-RAS testing in Treatment
Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic
Analysis.
OBJECTIVE: The objective of this systematic review is to determine the
predictive value of KRAS testing in the treatment of metastatic colorectal
cancer (mCRC) with two anti-EGFR agents, cetuximab and panitumumab. Economic
analyses are also being conducted to evaluate the cost-effectiveness of KRAS
testing.
CLINICAL NEED: CONDITION AND TARGET POPULATION Metastatic colorectal cancer
(mCRC) is usually defined as stage IV disease according to the American Joint
Committee on Cancer tumour node metastasis (TNM) system or stage D in the Duke's
classification system. Patients with advanced colorectal cancer (mCRC) either
present with metastatic disease or develop it through disease progression. KRAS
(Kristen-RAS, a member of the rat sarcoma virus (ras) gene family of oncogenes)
is frequently mutated in epithelial cancers such as colorectal cancer, with
mutations occurring in mutational hotspots (codons 12 and 13) of the KRAS
protein. Involved in EGFR-mediated signalling of cellular processes such as cell
proliferation, resistance to apoptosis, enhanced cell motility and
neoangiogenesis, a mutation in the KRAS gene is believed to be involved in
cancer pathogenesis. Such a mutation is also hypothesized to be involved in
resistance to targeted anti-EGFR (epidermal growth factor receptor with tyrosine
kinase activity) treatments such as cetuximab and panitumumab, hence, the
important in evaluating the evidence on the predictive value of KRAS testing in
this context. KRAS MUTATION TESTING IN ADVANCED COLORECTAL CANCER: Both
cetuximab and panitumumab are indicated by Health Canada in the treatment of
patients with metastatic colorectal cancer whose tumours are WT for the KRAS
gene. Cetuximab may be offered as monotherapy in patients intolerant to
irinotecan-based chemotherapy or in patients who have failed both irinotecan and
oxaliplatin-based regimens and who received a fluoropyrimidine. It can also be
administered in combination with irinotecan in patients refractory to other
irinotecan-based chemotherapy regimens. Panitumumab is only indicated as a
single agent after failure of fluoropyrimidine-, oxaliplatin-, and
irinotecan-containing chemotherapy regimens. In Ontario, patients with advanced
colorectal cancer who are refractory to chemotherapy may be offered the targeted
anti-EGFR treatments cetuximab or panitumumab. Eligibility for these treatments
is based on the KRAS status of their tumour, derived from tissue collected from
surgical or biopsy specimens. It is believed that KRAS status is not affected by
treatments, therefore, for patients for whom surgical tissue is available for
KRAS testing, additional biopsies prior to treatment with these targeted agents
is not necessary. For patients that have not undergone surgery or for whom
surgical tissue is not available, a biopsy of either the primary or metastatic
site is required to determine their KRAS status. This is possible as status at
the metastatic and primary tumour sites is considered to be similar.
RESEARCH QUESTION: To determine if there is predictive value of KRAS testing in
guiding treatment decisions with anti-EGFR targeted therapies in advanced
colorectal cancer patients refractory to chemotherapy.
LITERATURE SEARCH: The Medical Advisory Secretariat followed its standard
procedures and on May 18, 2010, searched the following electronic databases:
Ovid MEDLINE, EMBASE, Ovid MEDLINE In-Process & Other Non-Indexed Citations,
Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic
Reviews and The International Network of Agencies for Health Technology
Assessment database. The subject headings and keywords searched included
colorectal cancer, cetuximab, panitumumab, and KRAS testing. The search was
further restricted to English-language articles published between January 1,
2009 and May 18, 2010 resulting in 1335 articles for review. Excluded were case
reports, comments, editorials, nonsystematic reviews, and letters. Studies
published from January 1, 2005 to December 31, 2008 were identified in a health
technology assessment conducted by the Agency for Healthcare Research and
Quality (AHRQ), published in 2010. In total, 14 observational studies were
identified for inclusion in this EBA: 4 for cetuximab monotherapy, 7 for the
cetuximab-irinotecan combination therapy, and 3 to be included in the review for
panitumumab monotherapy
INCLUSION CRITERIA: English-language articles, and English or French-language
HTAs published from January 2005 to May 2010, inclusive.Randomized controlled
trials (RCTs) or observational studies, including single arm treatment studies
that include KRAS testing.Studies with data on main outcomes of interest,
overall and progression-free survival.Studies of third line treatment with
cetuximab or panitumumab in patients with advanced colorectal cancer refractory
to chemotherapy.For the cetuximab-irinotecan evaluation, studies in which at
least 70% of patients in the study received this combination therapy.
EXCLUSION CRITERIA: Studies whose entire sample was included in subsequent
publications which have been included in this EBA.Studies in pediatric
populations.Case reports, comments, editorials, or letters.
OUTCOMES OF INTEREST: Overall survival (OS), medianProgression-free-survival
(PFS), median.Response rates.Adverse event rates.Quality of life (QOL). SUMMARY
OF FINDINGS OF SYSTEMATIC REVIEW: CETUXIMAB OR PANITUMUMAB MONOTHERAPY: Based on
moderate GRADE observational evidence, there is improvement in PFS and OS
favouring patients without the KRAS mutation (KRAS wildtype, or KRAS WT)
compared to those with the mutation. CETUXIMAB-IRINOTECAN COMBINATION THERAPY:
There is low GRADE evidence that testing for KRAS may optimize survival benefits
in patients without the KRAS mutation (KRAS wildtype, or KRAS WT) compared to
those with the mutation. However, cetuximab-irinotecan combination treatments
based on KRAS status discount any effect of cetuximab in possibly reversing
resistance to irinotecan in patients with the mutation, as observed effects were
lower than for patients without the mutation. Clinical experts have raised
concerns about the biological plausibility of this observation and this
conclusion would, therefore, be regarded as hypothesis generating.
ECONOMIC ANALYSIS: Cost-effectiveness and budget impact analyses were conducted
incorporating estimates of effectiveness from this systematic review. Evaluation
of relative cost-effectiveness, based on a decision-analytic cost-utility
analysis, assessed testing for KRAS genetic mutations versus no testing in the
context of treatment with cetuximab monotherapy, panitumumab monotherapy,
cetuximab in combination with irinotecan, and best supportive care. Of
importance to note is that the cost-effectiveness analysis focused on the impact
of testing for KRAS mutations compared to no testing in the context of different
treatment options, and does not assess the cost-effectiveness of the drug
treatments alone.
CONCLUSIONS: KRAS status is predictive of outcomes in cetuximab and panitumumab
monotherapy, and in cetuximab-irinotecan combination therapy. While KRAS testing
is cost-effective for all strategies considered, it is not equally
cost-effective for all treatment options. Molecular diagnostics of human cancers may increase accuracy in prognosis,
facilitate the selection of the optimal therapeutic regimen, improve patient
outcome, reduce costs of treatment and favour development of personalized
approaches to patient care. Moreover sensitivity and specificity are fundamental
characteristics of any diagnostic method. We developed a highly sensitive
microarray for the detection of common KRAS and BRAF oncogenic mutations. In
colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster
of patients that does not respond to anti-EGFR therapies; the identification of
these mutations is therefore clinically extremely important. To verify the
technical characteristics of the microarray system for the correct
identification of the KRAS mutational status at the two hotspot codons 12 and 13
and of the BRAF(V600E) mutation in colorectal tumor, we selected 75 samples
previously characterized by conventional and CO-amplification at Lower
Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting
analysis and direct sequencing. Among these samples, 60 were collected during
surgery and immediately steeped in RNAlater while the 15 remainders were
formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the
proposed method was different for the 7 KRAS mutations tested and for the V600E
BRAF mutation. In particular, the microarray system has been able to detect a
minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A
blind validation displayed complete concordance of results. The excellent
agreement of the results showed that the new microarray substrate is highly
specific in assigning the correct genotype without any enrichment strategy. |
Which intraflagellar transport (IFT) motor protein has been linked to human skeletal ciliopathies? | Cytoplasmic dynein-2 (dynein-2) performs intraflagellar transport and is associated with human skeletal ciliopathies | Intraflagellar transport (IFT) depends on two evolutionarily conserved modules,
subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintece.
All six IFT-A components and their motor protein, DYNC2H1, have been linked to
human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD;
also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino
syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the
exception of IFT80, have unknown roles in human disease. To identify additional
IFT-B components defective in ciliopathies, we independently performed different
mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in
1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome
resequencing in 63 individuals with ATD. We thereby detected biallelic mutations
in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals
displayed abnormalities of the thorax and/or long bones, as well as renal,
hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS.
Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert
syndrome was present in 2 out of 12 families. Fibroblasts from affected
individuals showed disturbed ciliary composition, suggesting alteration of
ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated
the human phenotype and demonstrated a genetic interaction between ift172 and
ift80. In summary, we have identified defects in IFT172 as a cause of complex
ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an
additional IFT-B component, IFT172, similar to what has been shown for IFT-A. Bidirectional (anterograde and retrograde) motor-based intraflagellar transport
(IFT) governs cargo transport and delivery processes that are essential for
primary cilia growth and maintece and for hedgehog signaling functions. The
IFT dynein-2 motor complex that regulates ciliary retrograde protein transport
contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other
less well functionally defined subunits. Deficiency of IFT proteins, including
DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome
sequencing and a targeted next-generation sequencing panel, we identified a
total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of
Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40
repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein
intermediate chain associated with the retrograde intraflagellar transport
motor. Three-dimensional protein modeling suggests that the identified mutations
all affect residues critical for WDR34 protein-protein interactions. We find
that WDR34 concentrates around the centrioles and basal bodies in mammalian
cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the
dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests
that WDR34 could represent a previously unrecognized link between the
cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that
WDR34 is critical for ciliary functions essential to normal development and
survival, most probably as a previously unrecognized component of the mammalian
dynein-IFT machinery. Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT),
and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we
define for the first time the composition of the human cytoplasmic dynein-2
complex. We show that the proteins encoded by the ciliopathy genes WDR34 and
WDR60 are bona fide dynein-2 intermediate chains and are both required for
dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light
chain that is itself required for cilia function. We define several subunits
common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1)
and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and
roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1
and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as
it does with dynein-1. By contrast, the common dynein-1 regulators dynactin,
LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with
dynein-2. These data explain why mutations in either WDR34 or WDR60 cause
disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene. Members of the dynein family, consisting of cytoplasmic and axonemal isoforms,
are motors that move towards the minus ends of microtubules. Cytoplasmic
dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport, and is
implicated in viral infections and neurodegenerative diseases. Cytoplasmic
dynein-2 (dynein-2) performs intraflagellar transport and is associated with
human skeletal ciliopathies. Dyneins share a conserved motor domain that couples
cycles of ATP hydrolysis with conformational changes to produce movement. Here
we present the crystal structure of the human cytoplasmic dynein-2 motor bound
to the ATP-hydrolysis transition state analogue ADP.vanadate. The structure
reveals a closure of the motor's ring of six AAA+ domains (ATPases associated
with various cellular activites: AAA1-AAA6). This induces a steric clash with
the linker, the key element for the generation of movement, driving it into a
conformation that is primed to produce force. Ring closure also changes the
interface between the stalk and buttress coiled-coil extensions of the motor
domain. This drives helix sliding in the stalk which causes the microtubule
binding domain at its tip to release from the microtubule. Our structure answers
the key questions of how ATP hydrolysis leads to linker remodelling and
microtubule affinity regulation. |
What is the mechanism of action of ocrelizumab for treatment of multiple sclerosis? | Ocrelizumab is a cytolytic monoclonal antibody that binds CD20 antigen present of B cells. It is approved for treatment of multiple sclerosis. | Biogen Idec Inc, Genentech Inc, Roche Holding AG and Chugai Pharmaceutical Co
Ltd are developing ocrelizumab, a humanized mAb against CD20, for the potential
treatment of inflammatory disorders and B-cell maligcies. Ocrelizumab is
undergoing phase III clinical trials for rheumatoid arthritis and lupus
nephritis, and phase II trials for multiple sclerosis and hematological cancer.
Previously, ocrelizumab was also being developed for the treatment of systemic
lupus erythematosus (SLE) and neuromyelitis optica; however, development for SLE
has been discontinued. No development has been reported for neuromyelitis optica
and as of January 2007, this indication had been removed from the company
pipeline. BACKGROUND: B lymphocytes are implicated in the pathogenesis of multiple
sclerosis. We aimed to assess efficacy and safety of two dose regimens of the
humanised anti-CD20 monoclonal antibody ocrelizumab in patients with
relapsing-remitting multiple sclerosis.
METHODS: We did a multicentre, randomised, parallel, double-blind,
placebo-controlled study involving 79 centres in 20 countries. Patients aged
18-55 years with relapsing-remitting multiple sclerosis were randomly assigned
(1:1:1:1) via an interactive voice response system to receive either placebo,
low-dose (600 mg) or high-dose (2000 mg) ocrelizumab in two doses on days 1 and
15, or intramuscular interferon beta-1a (30 μg) once a week. The randomisation
list was not disclosed to the study centres, monitors, project statisticians or
to the project team at Roche. All groups were double blinded to group
assignment, except the interferon beta-1a group who were rater masked. At week
24, patients in the initial placebo, 600 mg ocrelizumab, and interferon beta-1a
groups received ocrelizumab 600 mg; the 2000 mg group received 1000 mg. Our
primary endpoint was the total number of gadolinium-enhancing lesions (GEL) and
T1-weighted MRI at weeks 12, 16, 20, and 24. Analyses were done on an
intention-to-treat basis. This trial is registered with ClinicalTrials.gov,
number NCT00676715.
FINDINGS: 218 (99%) of the 220 randomised patients received at least one dose of
ocrelizumab, 204 (93%) completed 24 weeks of the study and 196 (89%) completed
48 weeks. In the intention-to-treat population of 218 patients, at week 24, the
number of gadolinium-enhancing lesions was 89% (95% CI 68-97; p<0·0001) lower in
the 600 mg ocrelizumab group than in the placebo group, and 96% (89-99;
p<0·0001) lower in the 2000 mg group. In exploratory analyses, both 600 mg and
2000 mg ocrelizumab groups were better than interferon beta-1a for GEL
reduction. We noted serious adverse events in two of 54 (4%; 95% CI 3·0-4·4)
patients in the placebo group, one of 55 (2%; 1·3-2·3) in the 600 mg ocrelizumab
group, three of 55 (5%; 4·6-6·3) in the 2000 mg group, and two of 54 (4%;
3·0-4·4) in the interferon beta-1a group.
INTERPRETATION: The similarly pronounced effects of B-cell depletion with both
ocrelizumab doses on MRI and relapse-related outcomes support a role for B-cells
in disease pathogenesis and warrant further assessment in large, long-term
trials.
FUNDING: F Hoffmann-La Roche Ltd, Biogen Idec Inc. Multiple sclerosis (MS) is an inflammatory and degenerative disease leading to
demyelination and axonal damage in the CNS. Autoimmunity plays a central role in
MS pathogenesis. Per definition, monoclonal antibodies are recombit
biological compounds with a well defined target, thus carrying the promise of
targeting pathogenic cells or molecules with high specificity, avoiding
undesired off-target effects. Natalizumab was the first monoclonal antibody to
be approved for the treatment of MS. Several other monoclonal antibodies are in
development and have demonstrated promising efficacy in phase II studies. They
can be categorized according to their mode of action into compounds targeting
(i) leukocyte migration into the CNS (natalizumab); (ii) cytolytic antibodies
(rituximab, ocrelizumab, ofatumumab, alemtuzumab); or (iii) antibodies and
recombit proteins targeting cytokines and chemokines and their receptors
(daclizumab, ustekinumab, atacicept, tabalumab [Ly-2127399], secukinumab
[AIN457]). In this review, we discuss the specific molecular targets, clinical
efficacy and safety of these compounds and discuss criteria to anticipate the
position of monoclonal antibodies in the diversifying armamentarium of MS
therapy in the coming years. Recent years have broadened the spectrum of therapeutic strategies and specific
agents for treatment of multiple sclerosis (MS). While immune-modulating drugs
remain the first-line agents for MS predomitly due to their benign safety
profile, our growing understanding of key processes in initiation and
progression of MS has pioneered development of new agents with specific targets.
One concept of these novel drugs is to hamper migration of immune cells towards
the affected central nervous system (CNS). The first oral drug approved for MS
therapy, fingolimod inhibits egress of lymphocytes from lymph nodes; the
monoclonal antibody natalizumab prevents inflammatory CNS infiltration by
blocking required adhesion molecules. The second concept is to deplete T cells
and/or B cells from the peripheral circulation using highly specific monoclonal
antibodies such as alemtuzumab (anti-CD52) or rituximab/ocrelizumab (anti-CD20).
All of these novel, highly effective agents are a substantial improvement in our
therapeutic armamentarium; however, they have in common to potentially lower the
abundance of immune cells within the CNS, thereby collaterally affecting immune
surveillance within this well-controlled compartment. In this review, we aim to
critically evaluate the risk/benefit ratio of therapeutic strategies in
treatment of MS with a specific focus on infectious neurological side effects. Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the
central nervous system considered the second cause of disability in young
adults. The prognosis of MS has improved significantly since the approval of the
first interferon β in 1993 but, compared to other diseases, few new therapeutic
products have been commercialized in the last years. However, currently, there
are more than 600 ongoing clinical trials and new drugs that aim to improve
efficacy and a more convenient schedule of administration, will appear shortly
on the market. On the other hand, new safety issues will arise as well as a
significant economic impact on the health system. The main efficacy and safety
results of these drugs are reviewed in this paper. They can be classified into 2
groups: oral (fingolimod, laquinimod, teriflunomide, BG-12 [dimethyl fumarate],
oral cladribine, dalfampridine) and monoclonal antibodies (rituximab,
ocrelizumab, ofatumumab, daclizumab, alemtuzumab). The continuous improvements of our understanding of the pathophysiological
changes that occur in multiple sclerosis (MS) have translated into many novel
therapeutic agents at different stages of development. These agents target more
specifically the innate or the adaptive immune response. We will review agents
available or under development that target the humoral pathways of the adaptive
immune response. As such, humoral targeted immunotherapies that are being
developed for MS are discussed herein: rituximab, ocrelizumab, and ofatumumab
show promise as B-cell depleting agents. Other agents, such as atacicept were
suspended during development in MS due to increased inflammatory activity versus
the placebo. Although most agents were tested in relapsing-remitting forms of
MS, rituximab and ocrelizumab have both been studied in progressive MS, whereas
ocrelizumab only is currently moving forward in primary progressive MS trials.
We provide an overview of agents available and under development that target the
humoral response and include their mechanisms of action, safety profiles, and
results of clinical trials. Drug development for multiple sclerosis (MS), as with any other neurological
disease, faces numerous challenges, with many drugs failing at various stages of
development. The disease-modifying therapies (DMTs) first introduced for MS are
only moderately effective, but given the lack of competition, they have been
widely accepted in clinical practice. Although safety and efficacy continue to
be the two main metrics by which drugs will be judged, the newer agents in the
market also face challenges of a more comparative nature-are they more
efficacious than the currently available drugs on the market? Are they safer or
better tolerated? Do they offer any practical advantages over current
treatments? Fingolimod represented a milestone following its approval as an oral
drug for MS in 2010, offering patients a far more convenient administration
route. However, association with cardiovascular complications has led to a more
cautious approach in its initial prescribing, now requiring cardiac monitoring
for the first 6 h as well as subsequent monitoring of blood pressure and for
macular oedema. Natalizumab, amongst licensed drugs, represents the current
benchmark for efficacy. The risk of progressive multifocal leukoencephalopathy
during natalizumab treatment is now more quantifiable. Other monoclonal
antibodies are in various phases of development. Marketing authorisation for
alemtuzumab has been filed, and whilst trial data suggest that its efficacy
outperforms both licensed drugs and others in development, there is a
significant risk of secondary autoimmunity. Its once-yearly administration,
however, seems particularly advantageous. Rituximab is unlikely to be developed
further as its license will expire, but ocrelizumab, another monoclonal antibody
directly targeting B cells, is currently in phase 2 development and looks
promising. Daclizumab is also moderately efficacious but may struggle to
establish itself given its monthly subcutaneous dosing. There are new oral drugs
in development, and it is likely that BG-12 will be licensed this year. This has
been licensed for psoriasis so there are good safety data in humans that may
also hold true in MS; however, its three times daily dosage will probably impact
on patient compliance. Laquinimod has lower efficacy than BG-12 but appears safe
and could find a place as a first-line agent. Teriflunomide has just been
licensed by the US FDA and may challenge the current injectable first-line
therapies as it has a similar efficacy but the advantage of being taken orally.
However, risk of teratogenicity may caution against its use in some women of
child-bearing potential. This review will examine drugs that have been recently
approved as well as those that are in late phase 2 or 3 development as treatment
for relapsing MS, highlighting their mechanism of action as well as the clinical
trial and safety data before discussing their potential for success in an
increasingly florid and complex DMT armamentarium. Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines
in medicine, such as oncology, rheumatology, gastroenterology, dermatology and
transplant rejection prevention. Since the introduction and reintroduction of
the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained
relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs
have been tested in clinical trials in relapsing-remitting MS, and in
progressive forms of MS. One of the agents that might soon be approved for very
active forms of relapsing-remitting MS is alemtuzumab, a humanized mAb against
CD52. This review provides insights into clinical studies with the mAbs
natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. Multiple sclerosis (MS) and chronic inflammatory demyelinating
polyradiculoneuropathy (CIDP) represent chronic, autoimmune demyelinating
disorders of the central and peripheral nervous system. Although both disorders
share some fundamental pathogenic elements, treatments do not provide uniform
effects across both disorders. We aim at providing an overview of current and
future disease-modifying strategies in these disorders to demonstrate
communalities and distinctions. Intravenous immunoglobulins (IVIG) have
demonstrated short- and long-term beneficial effects in CIDP but are not
effective in MS. Dimethyl fumarate (BG-12), teriflunomide and laquinimod are
orally administered immunomodulatory drugs that are already approved or likely
to be approved in the near future for the basic therapy of patients with
relapsing-remitting MS (RRMS) due to positive results in Phase III clinical
trials. However, clinical trials with these drugs in CIDP have not (yet) been
initiated. Natalizumab and fingolimod are approved for the treatment of RRMS,
and trials to evaluate their safety and efficacy in CIDP are now planned.
Alemtuzumab, ocrelizumab and daclizumab respresent monoclonal antibodies in
advanced stages of clinical development for their use in RRMS patients. Attempts
to study the safety and efficacy of alemtuzumab and B cell-depleting anti-CD20
antibodies, i.e. rituximab, ocrelizumab or ofatumumab, in CIDP patients are
currently under way. We provide an overview of the mechanism of action and
clinical data available on disease-modifying immunotherapy options for MS and
CIDP. Enhanced understanding of the relative effects of therapies in these two
disorders may aid rational treatment selection and the development of innovative
treatment approaches in the future. Multiple sclerosis (MS) is a neurodegenerative disease with a major inflammatory
component that constitutes the most common progressive and disabling
neurological condition in young adults. Injectable immunomodulatory medicines
such as interferon drugs and glatiramer acetate have dominated the MS market for
over the past two decades but this situation is set to change. This is because
of: (i) patent expirations, (ii) the introduction of natalizumab, which targets
the interaction between leukocytes and the blood-CNS barrier, (iii) the launch
of three oral immunomodulatory drugs (fingolimod, dimethyl fumarate and
teriflunomide), with another (laquinimod) under regulatory review and (iv) a
number of immunomodulatory monoclonal antibodies (alemtuzumab, daclizumab and
ocrelizumab) about to enter the market. Current and emerging medicines are
reviewed and their impact on people with MS considered. Multiple sclerosis (MS) is a presumed autoimmune disorder of the central nervous
system, resulting in inflammatory demyelination and axonal and neuronal injury.
New diagnostic criteria that incorporate magnetic resoce imaging have
resulted in earlier and more accurate diagnosis of MS. Several immunomodulatory
and immunosuppressive therapeutic agents are available for relapsing forms of
MS, which allow individualized treatment based upon the benefits and risks.
Disease-modifying therapies introduced in the 1990s, the beta-interferons and
glatiramer acetate, have an established track record of efficacy and safety,
although they require administration via injection. More recently, monoclonal
antibodies have been engineered to act through specific mechanisms such as
blocking alpha-4 integrin interactions (natalizumab) or lysing cells bearing
specific markers, for example CD52 (alemtuzumab) or CD20 (ocrelizumab and
ofatumumab). These agents can be highly efficacious, but sometimes have serious
potential complications (natalizumab is associated with progressive multifocal
leukoencephalopathy; alemtuzumab is associated with the development of new
autoimmune disorders). Three new oral therapies (fingolimod, teriflunomide and
dimethyl fumarate, approved for MS treatment from 2010 onwards) provide
efficacy, tolerability and convenience; however, as yet, there are no long-term
postmarketing efficacy and safety data in a general MS population. Because of
this lack of long-term data, in some cases, therapy is currently initiated with
the older, safer injectable medications, but patients are monitored closely with
the plan to switch therapies if there is any indication of a suboptimal response
or intolerance or lack of adherence to the initial therapy. For patients with MS
who present with highly inflammatory and potentially aggressive disease, the
benefit-to-risk ratio may support initiating therapy using a drug with greater
potential efficacy despite greater risks (e.g. fingolimod or natalizumab if JC
virus antibody-negative). The aim of this review is to discuss the clinical
benefits, mechanisms of action, safety profiles and monitoring strategies of
current MS disease-modifying therapies in clinical practice and of those
expected to enter the market in the near future. INTRODUCTION: Monoclonal antibodies play an important role in the therapy of
different autoimmune diseases. With the introduction of natalizumab, the
importance of monoclonal antibodies in the therapy of multiple sclerosis (MS)
has dramatically increased during the past years.
AREAS COVERED: In this review, we will focus on newly approved and emerging
antibodies for MS therapy. Based on published original articles and citable
meeting abstracts, we will discuss their mode of action as well as data on
efficacy and safety.
EXPERT OPINION: Natalizumab was a breakthrough in MS therapy. However, side
effects of this monoclonal antibody limit its use. The risk/benefit ratios of
new biologicals in MS therapy are not yet clear. High-yield process daclizumab
might qualify as first-line MS therapy, unless hepatotoxicity becomes a relevant
safety concern. Alemtuzumab has been approved for MS therapy in Europe but will
be reserved for selected patients with highly active disease due to frequent
induction of potentially dangerous secondary autoimmune phenomena. Ocrelizumab
will likely also be licensed as a second-line therapy in highly active MS.
Neutralizing antibodies to interleukin (IL)-17A and blocking antibodies to
leucine rich repeat and Ig domain containing 1 might be the most interesting
upcoming new antibodies as both offer a new and pathophysiologically relevant
approach in MS therapy. Rapid advances are occurring in multiple sclerosis disease modifying therapies.
Recent therapeutic advances include modifications to improve tolerability of
existing products (e.g. interferon beta and glatiramer acetate), development of
novel anti-neuroinflammatory medications (e.g. fingolimod, teriflunomide and
dimethyl fumarate, daclizumab, alemtuzumab, ocrelizumab) and investigation of
treatments in progressive MS (e.g. natalizumab, mastinib, natalizumab,
siponimod). The impact of vitamin D supplementation on the disease course in
relapsing MS patients is also being studied in several clinical trials. This
article reviews the current state of the field with a forward look to the next
phase of MS research that could focus on strategies to promote remyelination and
provide neuronal protection. B linage cells are versatile players in multiple sclerosis (MS) and
neuromyelitis optica/neuromyelitis optica spectrum disorder (NMO). New potential
targets of autoantibodies have been described recently. Pathogenic mechanisms
extend further to antigen presentation and cytokine production, which are
increasingly recognized as therapeutic targets. In addition to pro-inflammatory
effects of B cells, they may act also as anti-inflammatory via production of
interleukin (IL)-10, IL-35, and other mechanisms. Definition of regulatory B
cell subsets is an ongoing issue. Recent studies have provided evidence for a
loss of B cell self-tolerance in MS. An immunogenetic approach demonstrated
exchange of B cell clones between CSF and blood. The central nervous system
(CNS) of MS patients fosters B cell survival, at least partly via BAFF and
APRIL. The unexpected increase of relapses in a trial with a soluble BAFF/APRIL
receptor (atacicept) suggests that this system is involved in MS, but with
features that are not yet understood. In this review, we further discuss
evidence for B cell and Ig contribution to human MS and NMO pathogenesis,
pro-inflammatory and regulatory B cell effector functions, impaired B cell
immune tolerance, the B cell-fostering microenvironment in the CNS, and B
cell-targeted therapeutic interventions for MS and NMO, including CD20 depletion
(rituximab, ocrelizumab, and ofatumumab), anti-IL6-R (tocilizumab),
complement-blocking (eculizumab), inhibitors of AQP4-Ig binding (aquaporumab,
small molecular compounds), and BAFF/BAFF-R-targeting agents. In multiple sclerosis (MS), B cell-depleting therapy using monoclonal anti-CD20
Abs, including rituximab (RTX) and ocrelizumab, effectively reduces disease
activity. Based on indirect evidence, it is generally believed that elimination
of the Ag-presenting capabilities and Ag nonspecific immune functions of B cells
underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T
cells) was shown to also express CD20, but controversy prevails surrounding the
true existence of this T cell subpopulation. Using single-cell imaging flow
cytometry and expression profiling of sorted lymphocyte subsets, we
unequivocally demonstrate the existence of CD3(+)CD20(dim) T cells. We show that
in MS patients, increased levels of CD3(+)CD20(dim) T cells are effectively
depleted by RTX. The pathological relevance of this T cell subset in MS remains
to be determined. However, given their potential proinflammatory functionality,
depletion of CD20-expressing T cells may also contribute to the therapeutic
effect of RTX and other mAbs targeting CD20. Until the mid 1990s, with the appearance of interferon beta and glatiramer
acetate, there was no treatment for multiple sclerosis (MS). However, due to
their moderate therapeutic potential in some patients, a broad search was
continued to find new and more effective treatment strategies, largely
concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for
the treatment of MS, was approved at the end of 2004, representing a major
advance in the field of neuroimmunology. Today, there is broad experience with
natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab,
ofatumumab and anti-lingo-1) that are pending commercialization or are under
phase II or III of development with promising results. The present review
analyzes the efficacy and safety results of all these drugs. |
What is the link between Nonidet-40 (NP-40) and biotinylation? | 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins. | We describe here a simple procedure for greatly reducing contamination of
nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which
represent a major source of non-specific background when employing BirA-mediated
biotinylation tagging for the purification and characterization of nuclear
protein complexes by mass spectrometry. We show that the use of 0.5% of the
non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is
sufficient to practically eliminate contamination of nuclear extracts by
carboxylases and to greatly reduce background signals in downstream mass
spectrometric analyses. |
Which is the transcript responsible for X-chromosome inactivation? | The long non- coding RNA Xist (X inactive specific transcript) | During early mammalian embryogenesis, one of the two X chromosomes in somatic
cells of the female becomes inactivated through a process that is thought to
depend on a unique initiator region, the X-chromosome inactivation center (Xic).
The recently characterized Xist sequence (X-inactive-specific transcript) is
thought to be a possible candidate for Xic. In mice a further genetic element,
the X chromosome-controlling element (Xce), is also known to influence the
choice of which of the two X chromosomes is inactivated. We report that a region
of the mouse X chromosome lying 15 kb distal to Xist contains several sites that
show hypermethylation specifically associated with the active X chromosome.
Analysis of this region in various Xce strains has revealed a correlation
between the strength of the Xce allele carried and the methylation status of
this region. We propose that such a region could be involved in the initial
stages of the inactivation process and in particular in the choice of which of
the two X chromosomes present in a female cell will be inactivated. In the mouse, the activity of Sry (sex-determining gene on the Y chromosome)
initiates the transformation of the indifferent gonad into a testis. In humans,
a partial Xp21 duplication leads to the development of ovaries instead of testes
in XY individuals. This observation indicates that sex determination might also
be influenced by a gene-dosage compensation mechanism, in addition to a domit
action of the Sry gene. In female mammals, the regulation of X-linked gene
dosage at early embryogenesis is achieved through the inactivation of one of the
two X chromosomes. Here we have investigated the possibility that inactivation
of the X chromosome may play a role in male sex determination. We have shown,
using an X-linked lacZ transgenic mouse line, that loss of beta-galactosidase
activity occurs in certain somatic cells of the developing male urogenital
ridge. When changes associated with apoptosis of mesonephric tubules in the
developing urogenital ridges are taken into account, expression of the Xist (X
inactive specific transcript) gene correlates with X inactivation revealed by
loss of beta-galactosidase activity in very early mesonephric tubule epithelial
cells, gonadal interstitial mesenchymal cells and coelomic epithelial cells. The Xist (X inactive specific transcript) gene plays an essential role in X
chromosome inactivation. To elucidate the mechanisms controlling Xist expression
and X inactivation, we examined in vivo DNA-protein interactions in the Xist
promoter region in a female mouse cell line (BMSL2), which has distinguishable
Xist alleles. In vivo footprinting was accomplished by treatment of cells with
dimethyl sulfate or ultraviolet light, followed by ligation-mediated polymerase
chain reaction of purified DNA. The expressed allele on the inactive X
chromosome and the silent allele on the active X chromosome were separated by
the use of a restriction fragment length polymorphism prior to ligation-mediated
polymerase chain reaction. The chromatin structure of the Xist promoter was
found to be consistent with the activity state of the Xist gene. The silent
allele (on the active X chromosome) showed no footprints, while the expressed
allele (on the inactive X chromosome) showed footprints at a consensus sequence
for a CCAAT box, two weak Sp1 sites, and a weak TATA box. Recent years have seen rapid progress towards understanding the molecular
mechanisms involved in X chromosome inactivation (X inactivation). This progress
has largely revolved around the discovery of the X inactive specific transcript
(Xist) gene, which is known now to represent the master switch locus regulating
X inactivation. In adult cells Xist is transcribed exclusively from the inactive
X chromosome. The transcript has no apparent protein-coding potential and is
retained in the nucleus in close association with the domain occupied by the
inactive X chromosome. It is thus thought to represent a functional RNA molecule
which acts as the primary signal responsible for the propagation of X
inactivation. Developmental regulation of Xist correlates with the developmental
timing of X inactivation. Recent results have demonstrated that Xist is both
necessary and sufficient for X inactivation. Goals for the future are to
understand the mechanism of Xist regulation which underlies the establishment of
appropriate X inactivation patterns and to determine how Xist RNA participates
in the process of propagating inactivation in cis. Expression of the X inactive-specific transcript (Xist) is thought to be
essential for the initiation of X chromosome inactivation and dosage
compensation during female embryo development. In the present study, we analyzed
the patterns of Xist transcription and the onset of X chromosome inactivation in
bovine preattachment embryos. Reverse transcription-polymerase chain reaction
(RT-PCR) revealed the presence of Xist transcripts in all adult female somatic
tissues evaluated. In contrast, among the male tissues examined, Xist expression
was detected only in testis. No evidence for Xist transcription was observed
after a single round of RT-PCR from pools of in vitro-derived embryos at the 2-
to 4-cell stage. Xist transcripts were detected as a faint amplicon at the
8-cell stage initially, and consistently thereafter in all stages examined up to
and including the expanded blastocyst stage. Xist transcripts, however, were
subsequently detected from the 2-cell stage onward after nested RT-PCR.
Preferential [3H]thymidine labeling indicative of late replication of one of the
X chromosomes was noted in female embryos of different developmental ages as
follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11
(72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts. These
results suggest that Xist expression precedes the onset of late replication in
the bovine embryo, in a pattern compatible with a possible role of bovine Xist
in the initiation of X chromosome inactivation. Initiation of X chromosome inactivation requires the presence, in cis, of the X
inactivation center (XIC). The Xist gene, which lies within the XIC region in
both human and mouse and has the unique property of being expressed only from
the inactive X chromosome in female somatic cells, is known to be essential for
X inactivation based on targeted deletions in the mouse. Although our
understanding of the developmental regulation and function of the mouse Xist
gene has progressed rapidly, less is known about its human homolog. To address
this and to assess the cross-species conservation of X inactivation, a 480-kb
yeast artificial chromosome containing the human XIST gene was introduced into
mouse embryonic stem (ES) cells. The human XIST transcript was expressed and
could coat the mouse autosome from which it was transcribed, indicating that the
factors required for cis association are conserved in mouse ES cells. Cis
inactivation as a result of human XIST expression was found in only a proportion
of differentiated cells, suggesting that the events downstream of XIST RNA
coating that culminate in stable inactivation may require species-specific
factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in
vitro differentiation, in contrast to the behavior of the mouse Xist gene in
undifferentiated ES cells, where an unstable transcript and no chromosome
coating are found. This may not only reflect important species differences in
Xist regulation but also provides evidence that factors implicated in Xist RNA
chromosome coating may already be present in undifferentiated ES cells. Equal expression of X-linked genes such as G6PD and PGK in females and males and
the initiation of X-chromosome inactivation are critically dependent on the
expression of the X-inactive specific transcript (Xist). The objective of the
present study was to determine the effects of in vitro production (IVP) and
nuclear transfer (NT) on the relative abundance (RA) of the X-linked transcripts
G6PD, PGK, and Xist in preimplantation bovine embryos. In experiment 1,
sex-determined IVP or in vivo-produced embryos were analyzed for mRNA expression
of the 3 genes. The sex ratio was 36% vs. 64% in IVP blastocysts and thus
deviated significantly from the expected ratio of 50% in the vivo control group.
The RA of G6PD transcripts was significantly higher in female IVP embryos than
in male embryos. In contrast, no significant differences were seen between in
vivo-derived female embryos and their male counterparts. At the morula stage,
female IVP embryos transcribed significantly more PGK mRNA than did male
embryos. However, blastocysts did not exhibit significant differences in PGK
transcripts. No differences were observed for in vivo-derived embryos with
regard to the RA of PGK transcripts. The RA of Xist mRNA was significantly
higher in all female embryos than in their male counterparts. In experiment 2,
IVP, in vivo-developed, NT-derived, and parthenogenetic embryos carrying two X
chromosomes of either maternal and paternal origin or of maternal origin only
(parthenogenotes) were analyzed for the RA of the 3 genes. In NT-derived
morulae, the RA of G6PD transcripts was significantly increased compared with
their IVP and in vivo-generated counterparts. G6PD transcript levels were
significantly increased in IVP blastocysts compared with in vivo-generated and
parthenogenetic embryos. At the morula stage, PGK transcripts were similar in
all groups, but the RA of PGK transcripts was significantly higher in IVP
blastocysts than in their in vivo-generated, parthenogenetic, and NT-derived
counterparts. The RA of Xist was significantly elevated in NT-derived morulae
compared with IVP, in vivo-generated, and parthenogenetic embryos. NT-derived
blastocysts showed an increased Xist expression compared with that of IVP, in
vivo-generated, and parthenogenetic embryos. Results of the present study show
for the first time that differences in X-chromosome-linked gene transcript
levels are related to a perturbed dosage compensation in female and male IVP and
female NT-derived embryos. This finding warrants further studies to improve IVP
systems and NT protocols to ensure the production of embryos with normal gene
expression patterns. Allelic expression differences contribute to phenotypic variation. In X
chromosome inactivation (XCI), unfavorable XCI ratios promote X-linked disease
penetrance in females. During XCI, one X is randomly silenced by Xist. X
chromosome choice is determined by asymmetric expression of Tsix whose antisense
action represses Xist. Here, we discover a cis element in the mouse
X-inactivation center that regulates Tsix. Xite harbors intergenic transcription
start sites and DNaseI hypersensitive sites with allelic differences. At the
onset of XCI, deleting Xite downregulates Tsix in cis and skews XCI ratios,
suggesting that Xite promotes Tsix persistence on the active X. Truncating Xite
RNA is inconsequential, indicating that Xite action does not require intact
transcripts. We propose that allele-specific Xite action promotes Tsix asymmetry
and generates X chromosome inequality. Therefore, Xite is a candidate for the
Xce, the classical modifier of XCI ratios. X chromosome inactivation begins when a novel chromosomal RNA (cRNA) from the
imprinted mouse Xist or human XIST locus coats or "paints" one X chromosome in
cis and initiates a cascade of chromosome remodeling events. Molecular
cytological studies have proven invaluable for understanding the distinctive
cellular behavior of this singular RNA involved in chromosome structure and
regulation. While the detailed mechanism of XIST/Xist (X-inactivation Specific
Transcript) RNA function remains largely unknown, recent advances provide new
insights into the complex cellular factors which impact the RNA's localization
to the chromosome, as well as the early events of chromosome remodeling that
follow painting by Xist RNA. Because chromatin changes can be directly
visualized on a silenced chromosome, X chromosome inactivation provides an
advantageous model to investigate genome-wide heterochromatin formation and
maintece, with wide-ranging implications for normal cells and disease. X inactivation in female mammals involves transcriptional silencing of an entire
chromosome in response to a cis-acting noncoding RNA, the X inactive-specific
transcript (Xist). Xist can also inactivate autosomal sequences, for example, in
X;autosome translocations; but here, silencing appears to be relatively
inefficient. This variation has been attributed to either attenuated spreading
of Xist RNA at the onset of X inactivation or inefficient maintece of
autosomal silencing. Evidence to date has favored the latter. Here, we
demonstrate attenuated spreading of Xist RNA at the onset of X inactivation in
the T(X;4)37H X;autosome translocation. Our findings provide direct evidence
that underlying chromosome/chromatin features can disrupt spreading of the
primary inactivating signal. X inactivation, the transcriptional silencing of one of the two X chromosomes in
female mammals, achieves dosage compensation of X-linked genes relative to XY
males. In eutherian mammals X inactivation is regulated by the X-inactive
specific transcript (Xist), a cis-acting non-coding RNA that triggers silencing
of the chromosome from which it is transcribed. Marsupial mammals also undergo X
inactivation but the mechanism is relatively poorly understood. We set out to
analyse the X chromosome in Monodelphis domestica and Didelphis virginiana,
focusing on characterizing the interval defined by the Chic1 and Slc16a2 genes
that in eutherians flank the Xist locus. The synteny of this region is retained
on chicken chromosome 4 where other loci belonging to the evolutionarily ancient
stratum of the human X chromosome, the so-called X conserved region (XCR), are
also located. We show that in both M. domestica and D. virginiana an
evolutionary breakpoint has separated the Chic1 and Slc16a2 loci. Detailed
analysis of opossum genomic sequences revealed linkage of Chic1 with the Lnx3
gene, recently proposed to be the evolutionary precursor of Xist, and Fip1, the
evolutionary precursor of Tsx, a gene located immediately downstream of Xist in
eutherians. We discuss these findings in relation to the evolution of Xist and X
inactivation in mammals. Numerous sense-antisense gene pairs have been discovered in various organisms.
Antisense genes play important roles in establishing parentally imprinted gene
expression patterns in mammals. Typically, protein-coding sense genes are
reciprocally regulated by their non-coding antisense partners. One example for
antisense regulation is the Xist (X-inactive specific transcript) and Tsix gene
pair, which is pivotal in X-inactivation. Xist works as a functional RNA
molecule that recruits repressive chromatin factors towards one of the female Xs
for inactivation. Antisense Tsix transcription negatively regulates Xist and
protects one X-chromosome in cis from inactivation by Xist. Albeit, the precise
molecular mechanism is still obscure it has been shown that Tsix transcription
regulates the chromatin structure by altering histone tail modifications and DNA
methylation at the Xist promoter. In addition, Xist and Tsix RNA form an RNA
duplexes in vivo and are processed to small RNAs, which have a potential
regulatory function. Here we review the latest findings and based on ample
experimental data consider models for antisense-mediated gene regulation in
X-inactivation. X chromosome inactivation (XCI), the silencing of one of the two X chromosomes
in XX female cells, equalises the dosage of X-linked genes relative to XY males.
The process is mediated by the non-coding RNA X inactive specific transcript
(Xist) that binds in cis and propagates along the inactive X chromosome elect,
triggering chromosome-wide silencing. The mechanisms by which Xist RNA binds and
spreads along the chromosome, and initiates Xist-mediated chromosome silencing
remain poorly understood. Accumulating evidence suggests that chromosome and
nuclear organisation are important in both processes. Notably, recent studies
have identified specific factors, previously shown to be components of the
nuclear matrix or scaffold, to play a role both in Xist RNA-binding and in
Xist-mediated silencing. In this review we provide a perspective on these
studies in the context of previous work on chromosome/nuclear architecture in
XCI. Random X chromosome inactivation (XCI), the eutherian mechanism of X-linked gene
dosage compensation, is controlled by a cis-acting locus termed the
X-inactivation center (Xic). One of the striking features that characterize the
Xic landscape is the abundance of loci transcribing non-coding RNAs (ncRNAs),
including Xist, the master regulator of the inactivation process. Recent
comparative genomic analyses have depicted the evolutionary scenario behind the
origin of the X-inactivation center, revealing that this locus evolved from a
region harboring protein-coding genes. During mammalian radiation, this
ancestral protein-coding region was disrupted in the marsupial group, whilst it
provided in eutherian lineage the starting material for the non-translated RNAs
of the X-inactivation center. The emergence of non-coding genes occurred by a
dual mechanism involving loss of protein-coding function of the pre-existing
genes and integration of different classes of mobile elements, some of which
modeled the structure and sequence of the non-coding genes in a species-specific
manner. The rising genes started to produce transcripts that acquired function
in regulating the epigenetic status of the X chromosome, as shown for Xist, its
antisense Tsix, Jpx, and recently suggested for Ftx. Thus, the appearance of the
Xic, which occurred after the divergence between eutherians and marsupials, was
the basis for the evolution of random X inactivation as a strategy to achieve
dosage compensation. X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X
inactivation specific transcript (Xist). The mechanism of chromosome-wide
silencing by Xist is poorly understood. While it is established that the 5'
region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a
conserved 26-mer sequence, is essential for Xist-mediated silencing,
high-resolution structural information for the A-repeats is not available. Here,
we report the three-dimensional solution structure of a 14-mer hairpin in the 5'
region of a human A-repeat. This hairpin is remarkably stable and adopts a novel
AUCG tetraloop fold, the integrity of which is required for silencing. We show
that, contrary to previous predictions, the 3' region of single or tandem
A-repeats mediates duplex formation in vitro. Significantly, mutations in this
region disrupt the inter-repeat duplex formation in vitro and abrogate the
silencing function of Xist A-repeats in vivo. Our data suggest that the complete
A-repeat region may be stabilized by inter-repeat duplex formation and, as such,
may provide a platform for multimerization and specific recognition of the AUCG
tetraloops by trans-acting factors. Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient.
Most SCNT-generated embryos die after implantation because of unidentified,
complex epigenetic errors in the process of postimplantation embryonic
development. Here we identify the most upstream level of dysfunction leading to
impaired development of clones by using RNAi against Xist, a gene responsible
for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA
into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist
expression at the morula stage, but failed to do so thereafter at the blastocyst
stage. However, we found that shortly after implantation, this aberrant XCI
status in cloned embryos had been corrected autonomously in both embryonic and
extraembryonic tissues, probably through a newly established XCI control for
postimplantation embryos. Embryo transfer experiments revealed that
siRNA-treated embryos showed 10 times higher survival than controls as early as
embryonic day 5.5 and this high survival persisted until term, resulting in a
remarkable improvement in cloning efficiency (12% vs. 1% in controls).
Importantly, unlike control clones, these Xist-siRNA clones at birth showed only
a limited dysregulation of their gene expression, indicating that correction of
Xist expression in preimplantation embryos had a long-term effect on their
postnatal normality. Thus, contrary to the general assumption, our results
suggest that the fate of cloned embryos is determined almost exclusively before
implantation by their XCI status. Furthermore, our strategy provides a promising
breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is
readily applicable to most mammal species. Mammalian dosage compensation requires silencing of one of the two X chromosomes
in females and is controlled by the X inactivation center (Xic). Xic contains
many of the regulatory elements for the mutual interplay of X-inactive specific
transcript (Xist) and Tsix, the antisense counterpart of Xist. The regulatory
elements control X chromosome inactivation (XCI) via the formation of DNA-DNA
and DNA-protein complexes with cis- and trans-acting factors. However, the
process-dependent regulation of Xist/Tsix by these elements in each XCI process
remains largely unknown. In this study, a 6-thioguanine-resistant female F(1)
hybrid mouse cell line (designated HOBMSKI2) was constructed from a cross
between a female HPRT-deficient transgenic mouse (designated BM3) and a male
wild type Mus spretus mouse (designated MS), which enabled the direct
discrimination of both allele-specific expression of X-linked genes and
allele-specific binding of proteins associated with XCI due to DNA polymorphisms
between BM3 and MS. Using this cell line, we found that Tsix on the active X
chromosome (Xa) was not expressed in somatic cells despite the fact that CTCF,
which ensures Tsix expression in embryonic stem cells, was still bound to the 5'
end of Tsix on Xa, implying that CTCF may function differently during each XCI
process and its trans-activating activity for Tsix expression may be lost in the
maintece process. In addition, the monoallelic expression of Tsix on Xa was
inhibited by epigenetic modification of the chromatin in the maintece
process, which was mediated by protein complexes recruited by MeCP2. The results
indicate the value of HOBMSKI2 in directly detecting the allele-specific binding
of CTCF and MeCP2 to the 5' end of Tsix. The HOBMSKI2 mouse line is a versatile
and useful resource for studying the molecular mechanism of the XCI process. The transcriptional silencing of one of the female X-chromosomes is a finely
regulated process that requires accumulation in cis of the long non-coding RNA
X-inactive-specific transcript (Xist) followed by a series of epigenetic
modifications. Little is known about the molecular machinery regulating
initiation and maintece of chromosomal silencing. Here, we introduce a new
version of our algorithm catRAPID to investigate Xist associations with a number
of proteins involved in epigenetic regulation, nuclear scaffolding,
transcription and splicing processes. Our method correctly identifies binding
regions and affinities of protein interactions, providing a powerful theoretical
framework for the study of X-chromosome inactivation and other events mediated
by ribonucleoprotein associations. An adjustment of sex ratio of offspring to the conditions present at conception
is seen in many mammals including horses. This depends on preferential survival
of male embryos under conditions of high energy intake. In several species,
growth factors including insulin like growth factor (IGF)-1 have been shown to
promote embryonic development by decreasing apoptosis and increasing cell
proliferation. We hypothesized that sex-related differences in IGF-1 expression
in equine embryos during the phase of maternal recognition of pregcy might
exist and thus contribute to preferential survival of embryos from either of
both sexes under specific environmental conditions. Insulin like growth factor-1
mRNA expression of in vivo-produced equine embryos on different days of
pregcy (Day 8, N = 6; Day 10, N = 8; Day 12, N = 14) was analyzed. Insulin
like growth factor-1 mRNA expression was evaluated by reverse transcription
quantitative polymerase chain reaction. The sex of the embryo was determined by
detection of X-inactivation specific transcript (Xist) RNA and equine sex
determining region of the Y chromosome DNA. Embryos positive for Xist expression
were classified as female, and Xist negative and equine sex determining region
of the Y chromosome positive embryos were classified as male. From 28 embryos
tested, 15 (54%) showed positive Xist expression and were thus classified as
female. Insulin like growth factor-1 mRNA expression was influenced by sex (P =
0.01) but not by day of pregcy (relative expression of IGF-1 in relation to
β-actin, Day 8: male 5.1 ± 2.1, female 11.4; Day 10: male 5.2 ± 1.6, female 17.4
± 6.7; Day 12: male 2.6 ± 0.3, female 11.6 ± 2.4). Results demonstrate an
increased expression of IGF-1 in female equine embryos. Sex-related influences
on expression of the IGF system are probably related to a gradual X chromosome
inactivation. Several studies have shown effects of diesel exhaust (DE) on the central nervous
system, but the mechanism is unclear. Fetal mice were exposed to whole DE
(contains gases and particles) in an inhalation chamber, and cerebrum gene
expression changes were examined by gene assay (microarray and quantitative
real-time PCR). By microarray, upregulation of Xist, B-raf and Drwms2 were
detected. Especially, mRNA expression of Xist was increased in a
concentration-dependent manner in male and female mice. Xist (X-inactive
specific transcript) is a major effector of the X-inactivation process, and
X-linked genes are highly expressed in brain tissue and consistent with a role
in brain developments. By quantitative real-time PCR, Tsix (crucial noncoding
antisense partner of Xist) and other X-linked genes (Mecp2, Hprt1, and Sts) were
examined; Tsix was upregulated, and other X-linked genes were unaffected in the
male and female mice. Our findings suggest that exposure to DE increases Xist
and Tsix gene expression in utero without influencing X-linked gene expression.
An examination of Xist gene expression changes may provide an important
biomarker for DE-induced effects. The possibility of avoiding X-chromosome
inactivation (XCI) mechanisms by minimizing exposure to DE is expected. XIST, a long non-coding RNA, plays an important role in triggering X chromosome
inactivation in eutherians, and is used extensively for qualifying stem cells
and cloned embryos. However, a porcine XIST has not yet been thoroughly
identified despite its biological importance in a wide variety of research
fields. Here, we present a full-length porcine XIST sequence assembled using
known sequences (GenBank), RNA-Seq data (NCBI SRA), and PCR/sequencing. The
proposed porcine XIST gene model encodes a 25,215-bp transcript consisting of 7
exons, including two conserved and two porcine-specific repeat regions.
Transcription covering the entire XIST region was observed specifically in
female cells, but not in male cells. We also identified eight transcription
starting sites (TSSs) and evaluated CpG methylation patterns in the upstream
(+2.0 kb) and downstream (-2.0 kb) regions. Sixty-seven CG di-nucleotides
identified in the target region were considered to be candidate CpG sites, and
were enriched in the following two regions: -284 to +53 bp (13 sites) and +285
to +1,727 bp (54 sites) from the selected TSS. Male 5` region of XIST (64.5
sites, 96.26%) had a higher level of CpG methylation than female DNA (33.4
sites, 49.85%). Taken together, our results revealed that the porcine XIST gene
is expressed exclusively in female cells, which is influenced by the lower level
of CpG methylation in the putative promoter region compared with male cells. The
porcine XIST presented in this study represents a useful tool for related
research areas such as porcine embryology and stem cell biology. |
What are viral vectors used for in optogenetics? | Viral vectors are used to express optogenetic constructs in selected cells. | We briefly review the current literature where optogenetics has been used to
study various aspects of astrocyte physiology in vitro and in vivo. This
includes both genetically engineered Ca(2+) sensors and effector proteins, such
as channelrhodopsin. We demonstrate how the ability to target astrocytes with
cell-specific viral vectors to express optogenetic constructs helped to unravel
some previously unsuspected roles of these inconspicuous cells. Optogenetics is a technique for controlling subpopulations of neurons in the
intact brain using light. This technique has the potential to enhance basic
systems neuroscience research and to inform the mechanisms and treatment of
brain injury and disease. Before launching large-scale primate studies, the
method needs to be further characterized and adapted for use in the primate
brain. We assessed the safety and efficiency of two viral vector systems
(lentivirus and adeno-associated virus), two human promoters (human synapsin
(hSyn) and human thymocyte-1 (hThy-1)) and three excitatory and inhibitory
mammalian codon-optimized opsins (channelrhodopsin-2, enhanced Natronomonas
pharaonis halorhodopsin and the step-function opsin), which we characterized
electrophysiologically, histologically and behaviorally in rhesus monkeys
(Macaca mulatta). We also introduced a new device for measuring in vivo
fluorescence over time, allowing minimally invasive assessment of construct
expression in the intact brain. We present a set of optogenetic tools designed
for optogenetic experiments in the non-human primate brain. Controlling activity of defined populations of neurons without affecting other
neurons in the brain is now possible by a new gene- and neuroengineering
technology termed optogenetics. Derived from microbial organisms, opsin genes
encoding light-activated ion channels and pumps (channelrhodopsin-2 [ChR2];
halorhodopsin [NpHR], respectively), engineered for expression in the mammalian
brain, can be genetically targeted into specific neural populations using viral
vectors. When exposed to light with appropriate wavelength, action potentials
can be triggered in ChR2-expressing neurons, whereas inhibition of action
potentials can be obtained in NpHR-expressing neurons, thus allowing for
powerful control of neural activity. Optogenetics is now intensively used in
laboratory animals, both in vitro and in vivo, for exploring functions of
complex neural circuits and information processing in the normal brain and
during various neurological conditions. The clinical perspectives of adopting
optogenetics as a novel treatment strategy for human neurological disorders have
generated considerable interest, largely because of the enormous potential
demonstrated in recent rodent and nonhuman primate studies. Restoration of
dopamine-related movement dysfunction in parkinsonian animals, amelioration of
blindness and recovery of breathing after spinal cord injury are a few examples
of such perspectives. Viral vector-mediated gene transfer has become increasingly valuable for primate
brain research, in particular for application of genetic methods (e.g.
optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and
infection patterns are viable options for obtaining viral vector-mediated
transgene delivery that is selective for particular neuronal pathways. For
example, several types of viral vectors can infect axon terminals (retrograde
infections), which enables targeted transgene delivery to neurons that directly
project to a particular viral injection region. Although recent studies in
rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9
(AAV9) efficiently transduce neurons, the tropisms and infection patterns remain
poorly understood in primate brains. Here, we constructed recombit AAV8 or
AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven
by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and
stereotaxically injected it into several brain regions in marmosets and macaque
monkeys. Immunohistochemical analyses revealed almost exclusive colocalization
of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific
marker, indicating endogenous neuronal tropism of AAV9, which was consistent
with our previous results utilizing AAV8. Injections of either AAV8-EGFP or
AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local
striatal neurons as a result of local infection, as well as expression in
dopaminergic neurons of the substantia nigra via retrograde transport along
nigrostriatal axonal projections. Retrograde infections were also observed in
the frontal cortex and thalamus, which are known to have direct projections to
the striatum. These local and retrograde gene transfers were further
demonstrated in the geniculocortical pathway of the marmoset visual system.
These findings indicate promising capabilities of AAV8 and AAV9 to deliver
molecular tools into a range of primate neural systems in pathway-specific
manners through their neuronal tropisms and infection patterns. Classically, temporally precise excitation of membrane potential in neurons
within intact tissue can be achieved by direct electrical stimulation or
indirect electrical stimulation induced by changing magnetic fields. Both of
these approaches have a predetermined selectivity based on the biophysical
properties of the nervous tissue and membrane in the region of the stimulation.
A recent advance in selective excitation of neurons is the "optogenetic"
approach utilizing channelrhodopsins (ChRs). By expressing the light-responsive
ChR in neurons using cell-type selective promoters or other methods, specific
neurons can be depolarized by light in a temporally precise manner with
millisecond resolution even if their membrane biophysical properties are less
favorable for electrical stimulation. In addition, ChRs can be used to
depolarize nonneuronal cells in the nervous tissue, and to sustain
depolarization over a prolonged period of time, both of which cannot be achieved
with electrical or magnetic stimulations. To conduct an experiment with ChR,
experimenters need to make the correct choices on the three main components to
such an experiment: the expression system, the illumination source, and the ChR
variant used. This chapter aims to provide some discussions on the current
developments of these aspects of the experiments. To express ChR in neurons, the
common expression systems include viral vectors, in utero electroporation, and
transgenic animals, each with their advantages and limitations regarding the
cost, expression pattern, and the required effort. In terms of the
instrumentation, an illumination source that is capable of providing the desired
wavelength with high intensity is crucial for the success of the experiment. The
important factors regarding the light source used include the cost, light
density output, efficiency for fiber coupling for in vivo rodent experiments,
and the available methods to control light intensity and onset/termination. The
third component of the experiment is the choice of the appropriate variants of
ChR. Many novel ChR variants with unique properties have been engineered, and it
can be difficult for the experimenters to choose the right variant with the
desired properties for their experiments, as some information necessary for the
experimenter to make the right selection is often incomplete or unavailable.
Currently, the available variants for neuroscientific research are wild-type
ChR2, ChR2+H134R, ChETA, VChR1, SFO, ChD, ChEF, ChIEF, ChRGR, CatCh, and TC. The
features and limitations of these different variants are presented here. Lastly,
this chapter will provide some suggestion for the future development of the
light source, expression system, and the development of the "next" generation of
ChRs. Understanding the structure and function of cortical circuits requires the
identification of and control over specific cell types in the cortex. To address
these obstacles, recent optogenetic approaches have been developed. The capacity
to activate, silence, or monitor specific cell types by combining genetics,
virology, and optics will decipher the role of specific groups of neurons within
circuits with a spatiotemporal resolution that overcomes standard approaches. In
this review, the various strategies for selective genetic targeting of a defined
neuronal population are discussed as well as the pros and cons of the use of
transgenic animals and recombit viral vectors for the expression of
transgenes in a specific set of neurons. Recent advances in optogenetic methods demonstrate the feasibility of selective
photoactivation at the soma of neurons that express channelrhodopsin-2 (ChR2),
but a comprehensive evaluation of different methods to selectively evoke
transmitter release from distant synapses using optogenetic approaches is
needed. Here we compared different lentiviral vectors, with
sub-population-specific and strong promoters, and transgenic methods to express
and photostimulate ChR2 in the long-range projections of paraventricular nucleus
of the hypothalamus (PVN) neurons to brain stem cardiac vagal neurons (CVNs).
Using PVN subpopulation-specific promoters for vasopressin and oxytocin, we were
able to depolarize the soma of these neurons upon photostimulation, but these
promoters were not strong enough to drive sufficient expression for optogenetic
stimulation and synaptic release from the distal axons. However, utilizing the
synapsin promoter photostimulation of distal PVN axons successfully evoked
glutamatergic excitatory post-synaptic currents in CVNs. Employing the Cre/loxP
system, using the Sim-1 Cre-driver mouse line, we found that the
Rosa-CAG-LSL-ChR2-EYFP Cre-responder mice expressed higher levels of ChR2 than
the Rosa-CAG-LSL-ChR2-tdTomato line in the PVN, judged by photo-evoked currents
at the soma. However, neither was able to drive sufficient expression to observe
and photostimulate the long-range projections to brainstem autonomic regions. We
conclude that a viral vector approach with a strong promoter is required for
successful optogenetic stimulation of distal axons to evoke transmitter release
in pre-autonomic PVN neurons. This approach can be very useful to study
important hypothalamus-brainstem connections, and can be easily modified to
selectively activate other long-range projections within the brain. The physiology and circuitry associated with dorsal cochlear nucleus neurons
(DCN) have been well described. The ability to remotely manipulate neuronal
activity in these neurons would represent a step forward in the ability to
understand the specific function of DCN neurons in hearing. Although,
optogenetics has been used to study the function of pathways in other systems
for several years, in the auditory system only neurons in the auditory cortex
have been studied using this technique. Adeno-associated viral vectors with
either channelrhodopsin-2 fused with GFP (ChR2-GFP) or halorhodopsin fused with
mCherry (HaloR-mCherry), capable of expressing light sensitive cation channels
or chloride pumps, respectively, were delivered into the dorsal cochlear nucleus
(DCN). One to 18 months later, expression of ChR2 and HaloR was observed
throughout the DCN. Rhodopsin distribution within the DCN was determined to be
within several cell types identified based on morphology and location within the
DCN. Expression of ChR2-GFP and HaloR-mCherry was found at both the injection
site as well as in regions receiving projections from the site. Wavelength
appropriate optical stimulation in vivo resulted in neuronal activity that was
significantly increased over pre-stimulation levels with no return to baseline
levels during the time of the light exposure. We also examined the effects of
optically driven neuronal activity on subsequent tone driven responses in the
DCN. In the DCN 75% of the 16 electrode sites showed decreased neuronal activity
in response to a tone immediately following light stimulation while six percent
were decreased following tone stimulation and 19% of the electrode sites showed
no change. This is in contrast to tone driven neuronal activity prior to the
light exposure in which the majority of electrode sites showed increased
neuronal activity. Our results indicate that expression and activation of
rhodopsin within neurons involved in auditory processing does not appear to have
deleterious effects on hearing even 18 months following expression. In addition,
virally targeted rhodopsins may be useful as tract tracers to delineate as well
as modulate the activity of pathways and specific neurons. In the future
rhodopsins can be targeted to specific subpopulations of auditory neurons.
Ultimately, photostimulation may provide a physiologically relevant method for
modulating the function of auditory neurons and affecting hearing outcomes. This
article is part of a Special Issue entitled Optogenetics (7th BRES). A critical technique for understanding how neuronal activity contributes to
behavior is determining whether perturbing it changes behavior. The advent of
optogenetic techniques allows the immediately reversible alteration of neuronal
activity in contrast to chemical approaches lasting minutes to hours.
Modification of behavior using optogenetics has had substantial success in
rodents but has not been as successful in monkeys. Here, we show how optogenetic
inactivation of superior colliculus neurons in awake monkeys leads to clear and
repeatable behavioral deficits in the metrics of saccadic eye movements. We used
our observations to evaluate principles governing the use of optogenetic
techniques in the study of the neuronal bases of behavior in monkeys,
particularly how experimental design must address relevant parameters, such as
the application of light to subcortical structures, the spread of viral
injections, and the extent of neuronal inactivation with light. Relapse to maladaptive eating habits during dieting is often provoked by stress.
Recently, we identified a role of dorsal medial prefrontal cortex (mPFC) neurons
in stress-induced reinstatement of palatable food seeking in male rats. It is
unknown whether endogenous neural activity in dorsal mPFC drives stress-induced
reinstatement in female rats. Here, we used an optogenetic approach, in which
female rats received bilateral dorsal mPFC microinjections of viral constructs
coding light-sensitive eNpHR3.0-eYFP or control eYFP protein and intracranial
fiber optic implants. Rats were food restricted and trained to lever press for
palatable food pellets. Subsequently, pellets were removed, and lever pressing
was extinguished; then the effect of bilateral dorsal mPFC light delivery on
reinstatement of food seeking was assessed after injections of the
pharmacological stressor yohimbine (an α-2 andrenoceptor antagonist) or pellet
priming, a manipulation known to provoke food seeking in hungry rats. Dorsal
mPFC light delivery attenuated yohimbine-induced reinstatement of food seeking
in eNpHR3.0-injected but not eYFP-injected rats. This optical manipulation had
no effect on pellet-priming-induced reinstatement or ongoing food-reinforced
responding. Dorsal mPFC light delivery attenuated yohimbine-induced Fos
immunoreactivity and disrupted neural activity during in vivo
electrophysiological recording in awake rats. Optical stimulation caused
significant outward currents and blocked electrically evoked action potentials
in eNpHR3.0-injected but not eYFP-injected mPFC hemispheres. Light delivery
alone caused no significant inflammatory response in mPFC. These findings
indicate that intracranial light delivery in eNpHR3.0 rats disrupts endogenous
dorsal mPFC neural activity that plays a role in stress-induced relapse to food
seeking in female rats. Optogenetics, the use of light to stimulate or inhibit neural circuits via viral
transduction of protein channels, has emerged as a possible method of treating
epilepsy. By introducing viral vectors carrying algal-derived cation or anion
channels, known as opsins, neurons that initiate or propagate seizures may be
silenced. To date, studies using this technique have been performed in animal
models, and current efforts are moving toward more sophisticated nonhuman
primate models. In this paper, the authors present a brief overview of the
development of optogenetics and review recent studies investigating optogenetic
modification of circuits involved in seizures. Further developments in the field
are explored, with an emphasis on how optogenetics may influence future
neurosurgical interventions. The dynamic nature of gene expression enables cellular programming, homeostasis
and environmental adaptation in living systems. Dissection of causal gene
functions in cellular and organismal processes therefore necessitates approaches
that enable spatially and temporally precise modulation of gene expression.
Recently, a variety of microbial and plant-derived light-sensitive proteins have
been engineered as optogenetic actuators, enabling high-precision spatiotemporal
control of many cellular functions. However, versatile and robust technologies
that enable optical modulation of transcription in the mammalian endogenous
genome remain elusive. Here we describe the development of light-inducible
transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating
the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2
protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not
require additional exogenous chemical cofactors, are easily customized to target
many endogenous genomic loci, and can be activated within minutes with
reversibility. LITEs can be packaged into viral vectors and genetically targeted
to probe specific cell populations. We have applied this system in primary mouse
neurons, as well as in the brain of freely behaving mice in vivo to mediate
reversible modulation of mammalian endogenous gene expression as well as
targeted epigenetic chromatin modifications. The LITE system establishes a novel
mode of optogenetic control of endogenous cellular processes and enables direct
testing of the causal roles of genetic and epigenetic regulation in normal
biological processes and disease states. Gene therapy in human disease has expanded rapidly in recent years with the
development of safer and more effective viral vectors, and presents a novel
approach to the treatment of epilepsy. Studies in animals models have
demonstrated that overexpression of inhibitory peptides can modify seizure
threshold, prevent the development of epilepsy, and modify established epilepsy.
More recently there has been a flurry of studies using optogenetics in which
light-activated channels expressed in neurons can transiently change neuronal
excitability on exposure to light, thereby enabling the development of closed
loop systems to detect and stop seizure activity. The treatment of status
epilepticus presents its own challenges. Because of both the delay in gene
expression following transfection and also the necessity of using focal
transfection, there are a limited number of situations in which gene therapy can
be used in status epilepticus. One such condition is epilepsia partialis
continua (EPC). We have used gene therapy in a model of EPC and have shown that
we can "cure" the condition. Recent evidence suggesting that gene therapy
targeting subcortical regions can modify generalized or more diffuse epilepsies,
indicates that the range of situations in status epilepticus in which gene
therapy could be used will expand. Tools enabling the manipulation of well defined neuronal subpopulations are
critical for probing complex neuronal networks. Cre recombinase (Cre) mouse
driver lines in combination with the Cre-dependent expression of proteins using
viral vectors--in particular, recombit adeno-associated viral vectors
(rAAVs)--have emerged as a widely used platform for achieving transgene
expression in specified neural populations. However, the ability of rAAVs to
further specify neuronal subsets on the basis of their anatomical connectivity
has been reported as limited or inconsistent. Here, we systematically tested a
variety of widely used neurotropic rAAVs for their ability to mediate retrograde
gene transduction in the mouse brain. We tested pseudotyped rAAVs of several
common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and
without Cre-dependent expression switches. Many of the rAAVs tested--in
particular, though not exclusively, Cre-dependent vectors--showed a robust
capacity for retrograde infection and transgene expression. Retrograde
expression was successful over distances as large as 6 mm and in multiple neuron
types, including olfactory projection neurons, neocortical pyramidal cells
projecting to distinct targets, and corticofugal and modulatory projection
neurons. Retrograde infection using transgenes such as ChR2 allowed for optical
control or optically assisted electrophysiological identification of neurons
defined genetically as well as by their projection target. These results
establish a widely accessible tool for achieving combinatorial specificity and
stable, long-term transgene expression to isolate precisely defined neuron
populations in the intact animal. The C1 neurons, located in the rostral ventrolateral medulla (VLM), are
activated by pain, hypotension, hypoglycemia, hypoxia, and infection, as well as
by psychological stress. Prior work has highlighted the ability of these neurons
to increase sympathetic tone, hence peripheral catecholamine release, probably
via their direct excitatory projections to sympathetic preganglionic neurons. In
this study, we use channelrhodopsin-2 (ChR2) optogenetics to test whether the C1
cells are also capable of broadly activating the brain's noradrenergic system.
We selectively expressed ChR2(H134R) in rostral VLM catecholaminergic neurons by
injecting Cre-dependent adeno-associated viral vectors into the brain of adult
dopamine-β-hydroxylase (DβH)(Cre/0) mice. Most ChR2-expressing VLM neurons (75%)
were immunoreactive for phenylethanolamine N-methyl transferease, thus were C1
cells, and most of the ChR2-positive axonal varicosities were immunoreactive for
vesicular glutamate transporter-2 (78%). We produced light microscopic evidence
that the axons of rostral VLM (RVLM) catecholaminergic neurons contact locus
coeruleus, A1, and A2 noradrenergic neurons, and ultrastructural evidence that
these contacts represent asymmetric synapses. Using optogenetics in tissue
slices, we show that RVLM catecholaminergic neurons activate the locus coeruleus
as well as A1 and A2 noradrenergic neurons monosynaptically by releasing
glutamate. In conclusion, activation of RVLM catecholaminergic neurons,
predomitly C1 cells, by somatic or psychological stresses has the potential
to increase the firing of both peripheral and central noradrenergic neurons. Many approaches that use viral vectors to deliver transgenes have limited
transduction efficiency yet require high levels of transgene expression. In
particular, infection via axon terminals is relatively inefficient but is a
powerful means of achieving infection of specific neuron types. Combining this
with optogenetic approaches requires high gene expression levels that are not
typically achieved with nontoxic retrogradely infecting vectors. We generated
rabies glycoprotein-pseudotyped lentiviral vectors that use a positive feedback
loop composed of a Tet promoter driving both its own tetracycline-dependent
transcription activator (tTA) ("TLoop") and channelrhodopsin-2-YFP (ChR2YFP). We
show that TLoop vectors strongly express proteins in a drug-controllable manner
in neurons that project to injection sites within the mouse brain. After initial
infection, the virus travels retrogradely, stably integrates into the host
genome, and expresses gene products. The expression is robust and allows
optogenetic studies of neurons projecting to the location of virus injection, as
demonstrated by fluorescence-targeted intracellular recordings. ChR2YFP
expression did not cause observable signs of toxicity and continued for up to 6
mo after infection. Expression can be reversibly blocked by administration of
doxycycline, if necessary, for expression of gene products that might be more
toxic. Overall, we present a system that will allow researchers to achieve high
levels of gene expression even in the face of inefficient viral transduction.
The particular vectors that we demonstrate may enhance efforts to gain a precise
understanding of the contributions of specific types of projection neurons to
brain function. Pontospinal noradrenergic neurons are thought to form part of a descending
endogenous analgesic system that exerts inhibitory influences on spinal
nociception. Using optogenetic targeting, we tested the hypothesis that
excitation of the locus ceruleus (LC) is antinociceptive. We transduced rat LC
neurons by direct injection of a lentiviral vector expressing channelrhodopsin2
under the control of the PRS promoter. Subsequent optoactivation of the LC
evoked repeatable, robust, antinociceptive (+4.7°C ± 1.0, p < 0.0001) or
pronociceptive (-4.4°C ± 0.7, p < 0.0001) changes in hindpaw thermal withdrawal
thresholds. Post hoc anatomical characterization of the distribution of
transduced somata referenced against the position of the optical fiber and
subsequent further functional analysis showed that antinociceptive actions were
evoked from a distinct, ventral subpopulation of LC neurons. Therefore, the LC
is capable of exerting potent, discrete, bidirectional influences on thermal
nociception that are produced by specific subpopulations of noradrenergic
neurons. This reflects an underlying functional heterogeneity of the influence
of the LC on the processing of nociceptive information. Optogenetics is an emerging technology for the manipulation and control of
excitable tissues, such as the brain and heart. As this technique requires the
genetic modification of cells in order to inscribe light sensitivity, for
cardiac applications, here we describe the process through which neonatal rat
ventricular myocytes are virally infected in vitro with channelrhodopsin-2
(ChR2). We also describe in detail the procedure for quantitatively determining
the optimal viral dosage, including instructions for patterning gene expression
in multicellular cardiomyocyte preparations (cardiac syncytia) to simulate
potential in vivo transgene distributions. Finally, we address optical actuation
of ChR2-transduced cells and means to measure their functional response to
light. Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have
been proposed to contribute to drug-mediated addictive behaviors. Here we have
used an optogenetic approach to examine the role of NAc medium spiny neurons
(MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral
sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2)
were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to
selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory
circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic
currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo
affected neither the initiation nor the expression of cocaine-induced behavioral
sensitization. However, photostimulation during the drug withdrawal period
attenuated expression of cocaine-induced behavioral sensitization. These results
show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and
may contribute to relapse after cessation of drug abuse. Author information:
(1)Allen Institute for Brain Science, 551 N 34(th) Street, Seattle, WA 98103,
USA.
(2)UCL Institute of Ophthalmology, University College London, 11-43 Bath Street,
London, EC1V 9EL, UK.
(3)MIT Media Lab and McGovern Institute, Massachusetts Institute of Technology,
20 Ames Street, Cambridge, MA 02139, USA.
(4)Brain Research Institute, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland.
(5)Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198,
Japan.
(6)Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University
Avenue, Toronto, ON M5G 1X5, Canada.
(7)UCL Institute of Ophthalmology, University College London, 11-43 Bath Street,
London, EC1V 9EL, UK; Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city,
Saitama 351-0198, Japan.
(8)Department of Physiology, Brain Health Research Centre, University of Otago,
PO Box 913, Dunedin 9054, New Zealand.
(9)The Division of Brain Sciences, Department of Medicine, Imperial College
London, 160 DuCane Road, London, W12 0NN, UK.
(10)Allen Institute for Brain Science, 551 N 34(th) Street, Seattle, WA 98103,
USA. Electronic address: [email protected]. PURPOSE OF REVIEW: In this review, we will discuss the recent developments in
optogenetics and their potential applications in ophthalmology to restore vision
in retinal degenerative diseases.
RECENT FINDINGS: In recent years, we have seen major advances in the field of
optogenetics, providing us with novel opsins for potential applications in the
retina. Microbial opsins with improved light sensitivity and red-shifted action
spectra allow optogenetic stimulation at light levels well below the safety
threshold in the human eye. In parallel, remarkable success in the development
of highly efficient viral vectors for ocular gene therapy led to new strategies
of using these novel optogenetic tools for vision restoration.
SUMMARY: These recent findings show that novel optogenetic tools and viral
vectors for ocular gene delivery are now available providing many opportunities
to develop potential optogenetic strategies for vision restoration. The present palette of opsin-based optogenetic tools lacks a light-gated
potassium (K(+)) channel desirable for silencing of excitable cells. Here, we
describe the construction of a blue-light-induced K(+) channel 1 (BLINK1)
engineered by fusing the plant LOV2-Jα photosensory module to the small viral
K(+) channel Kcv. BLINK1 exhibits biophysical features of Kcv, including K(+)
selectivity and high single-channel conductance but reversibly photoactivates in
blue light. Opening of BLINK1 channels hyperpolarizes the cell to the K(+)
equilibrium potential. Ectopic expression of BLINK1 reversibly inhibits the
escape response in light-exposed zebrafish larvae. BLINK1 therefore provides a
single-component optogenetic tool that can establish prolonged, physiological
hyperpolarization of cells at low light intensities. |
Which domain allowing self-association do exist in TDP-43 and FUS proteins? | PRION PROTEINS | The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus)
mutants of Arabidopsis exhibit features of light-grown seedlings when grown in
the dark. Cloning and biochemical analysis of COP9 have revealed that it is a
component of a multiprotein complex, the COP9 signalosome (previously known as
the COP9 complex). Here, we compare the immunoaffinity and the biochemical
purification of the COP9 signalosome from cauliflower and confirm its
eight-subunit composition. Molecular cloning of subunit 4 of the complex
revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a
gene that maps to chromosome 5, near the chromosomal location of the cop8 and
fus4 mutations. Genetic complementation tests showed that the cop8 and fus4
mutations define the same locus, now designated as COP8. Molecular analysis of
the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific
molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant
background resulted in complete rescue of the mutant phenotype. Thus, we
conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of
possible molecular interactions by using the yeast two-hybrid assay indicated
that COP8 is capable of strong self-association as well as interaction with
COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins
being previously defined subunits of the Arabidopsis COP9 signalosome. A
comparative sequence analysis indicated that COP8 is highly conserved among
multicellular eukaryotes and is also similar to a subunit of the 19S regulatory
particle of the 26S proteasome. Prions are proteins that access self-templating amyloid forms, which confer
phenotypic changes that can spread from individual to individual within or
between species. These infectious phenotypes can be beneficial, as with yeast
prions, or deleterious, as with mammalian prions that transmit spongiform
encephalopathies. However, the ability to form self-templating amyloid is not
unique to prion proteins. Diverse polypeptides that tend to populate
intrinsically unfolded states also form self-templating amyloid conformers that
are associated with devastating neurodegenerative disorders. Moreover, two
RNA-binding proteins, FUS and TDP-43, which form cytoplasmic aggregates in
amyotrophic lateral sclerosis, harbor a 'prion domain' similar to those found in
several yeast prion proteins. Can these proteins and the neurodegenerative
diseases to which they are linked become 'infectious' too? Here, we highlight
advances that define the transmissibility of amyloid forms connected with
Alzheimer's disease, Parkinson's disease and Huntington's disease. Collectively,
these findings suggest that amyloid conformers can spread from cell to cell
within the brains of afflicted individuals, thereby spreading the specific
neurodegenerative phenotypes distinctive to the protein being converted to
amyloid. Importantly, this transmissibility mandates a re-evaluation of emerging
neuronal graft and stem-cell therapies. In this Commentary, we suggest how these
treatments might be optimized to overcome the transmissible conformers that
confer neurodegeneration. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease
that preferentially targets motor neurons. It was recently found that domit
mutations in two related RNA-binding proteins, TDP-43 (43-kDa TAR DNA-binding
domain protein) and FUS/TLS (fused in sarcoma/translated in liposarcoma) cause a
subset of ALS. The convergent ALS phenotypes associated with TDP-43 and FUS/TLS
mutations are suggestive of a functional relationship; however, whether or not
TDP-43 and FUS/TLS operate in common biochemical pathways is not known. Here we
show that TDP-43 and FUS/TLS directly interact to form a complex at endogenous
expression levels in mammalian cells. Binding was mediated by an unstructured
TDP-43 C-terminal domain and occurred within the context of a 300-400-kDa
complex that also contained C-terminal cleavage products of TDP-43 linked to
neuropathology. TDP-43 C-terminal fragments were excluded from large molecular
mass TDP-43 ribonucleoprotein complexes but retained FUS/TLS binding activity.
The functional significance of TDP-43-FUS/TLS complexes was established by
showing that RNAi silencing of either TDP-43 or FUS/TLS reduced the expression
of histone deacetylase (HDAC) 6 mRNA. TDP-43 and FUS/TLS associated with HDAC6
mRNA in intact cells and in vitro, and competition experiments suggested that
the proteins occupy overlapping binding sites. The combined findings demonstrate
that TDP-43 and FUS/TLS form a functional complex in intact cells and suggest
that convergent ALS phenotypes associated with TDP-43 and FUS/TLS mutations may
reflect their participation in common biochemical processes. Parkinson's disease (PD) is a movement disorder that afflicts over one million
in the U.S.; amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is less
prevalent but also has a high incidence. The two disorders sometimes present
together, making a comparative study of interest. Both ALS and PD are
neurodegenerative diseases, and are characterized by the presence of
intraneuronal inclusions; however, different classes of neurons are affected and
the primary protein in the inclusions differs between the diseases, and in some
cases is different in distinct forms of the same disease. These observations
might suggest that the more general approach of proteostasis pathway alteration
would be a powerful one in treating these disorders. Examining results from
human genetics and studies in model organisms, as well as from biochemical and
biophysical characterization of the proteins involved in both diseases, we find
that most instances of PD can be considered as arising from the misfolding, and
self-association to a toxic species, of the small neuronal protein α-synuclein,
and that proteostasis strategies are likely to be of value for this disorder.
For ALS, the situation is much more complex and less clear-cut; the available
data are most consistent with a view that ALS may actually be a family of
disorders, presenting similarly but arising from distinct and nonoverlapping
causes, including mislocalization of some properly folded proteins and
derangement of RNA quality control pathways. Applying proteostasis approaches to
this disease may require rethinking or broadening the concept of what
proteostasis means. Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal
neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43
and FUS, that harbor prion-like domains, cause some forms of ALS. There are at
least 213 human proteins harboring RNA recognition motifs, including FUS and
TDP-43, raising the possibility that additional RNA-binding proteins might
contribute to ALS pathogenesis. We performed a systematic survey of these
proteins to find additional candidates similar to TDP-43 and FUS, followed by
bioinformatics to predict prion-like domains in a subset of them. We sequenced
one of these genes, TAF15, in patients with ALS and identified missense
variants, which were absent in a large number of healthy controls. These
disease-associated variants of TAF15 caused formation of cytoplasmic foci when
expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and
FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila,
with the ALS-linked variants having a more severe effect than wild type.
Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization
of TAF15 in motor neurons of patients with ALS. We propose that
aggregation-prone RNA-binding proteins might contribute very broadly to ALS
pathogenesis and the genes identified in our yeast functional screen, coupled
with prion-like domain prediction analysis, now provide a powerful resource to
facilitate ALS disease gene discovery. Prions are self-templating protein conformers that are naturally transmitted
between individuals and promote phenotypic change. In yeast, prion-encoded
phenotypes can be beneficial, neutral or deleterious depending upon genetic
background and environmental conditions. A distinctive and portable 'prion
domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies
the majority of yeast prion proteins. Deletion of this domain precludes
prionogenesis and appending this domain to reporter proteins can confer
prionogenicity. An algorithm designed to detect prion domains has successfully
identified 19 domains that can confer prion behavior. Scouring the human genome
with this algorithm enriches a select group of RNA-binding proteins harboring a
canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of
210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these
are in the top 60 prion candidates in the entire genome. Startlingly, these
RNA-binding prion candidates are inexorably emerging, one by one, in the
pathology and genetics of devastating neurodegenerative disorders, including:
amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with
ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's
disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing
prion candidates, form cytoplasmic inclusions in the degenerating motor neurons
of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently,
perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing
prion candidate, has been connected with ALS and FTLD-U. We strongly suspect
that we have now merely reached the tip of the iceberg. We predict that
additional RNA-binding prion candidates identified by our algorithm will soon
surface as genetic modifiers or causes of diverse neurodegenerative conditions.
Indeed, simple prion-like transfer mechanisms involving the prion domains of
RNA-binding proteins could underlie the classical non-cell-autonomous emanation
of neurodegenerative pathology from originating epicenters to neighboring
portions of the nervous system. This article is part of a Special Issue entitled
RNA-Binding Proteins. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease
affecting motor neurons. Mutations in related RNA-binding proteins TDP-43,
FUS/TLS and TAF15 have been connected to ALS. These three proteins share several
features, including the presence of a bioinformatics-predicted prion domain,
aggregation-prone nature in vitro and in vivo and toxic effects when expressed
in multiple model systems. Given these commonalities, we hypothesized that a
related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit
similar properties and therefore could contribute to disease. Here, we report an
analysis of EWSR1 in multiple functional assays, including mutational screening
in ALS patients and controls. We identified three missense variants in EWSR1 in
ALS patients, which were absent in a large number of healthy control
individuals. We show that disease-specific variants affect EWSR1 localization in
motor neurons. We also provide multiple independent lines of in vitro and in
vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15,
including aggregation-prone behavior in vitro and ability to confer
neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also
revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a
potential role for EWSR1 in ALS, provide a collection of functional assays to be
used to assess roles of additional RNA-binding proteins in disease and support
an emerging concept that a class of aggregation-prone RNA-binding proteins might
contribute broadly to ALS and related neurodegenerative diseases. Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or
Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and
several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive
inclusions in FTLD and ALS patients are consistently co-labeled with stress
granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is
currently still debated. We determined the requirements for SG recruitment of
FUS and TDP-43 and found that cytoplasmic mislocalization is a common
prerequisite for SG recruitment of FUS and TDP-43. For FUS, the
arginine-glycine-glycine zinc finger domain, which is the protein's main RNA
binding domain, is most important for SG recruitment, whereas the glycine-rich
domain and RNA recognition motif (RRM) domain have a minor contribution and the
glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the
C-terminal glycine-rich domain are required for SG localization. ALS-associated
point mutations located in the glycine-rich domain of TDP-43 do not affect SG
recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is
enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails
to be recruited into SG. Consistently, inclusions in the cortex of FTLD
patients, which are enriched for C-terminal fragments, are not co-labeled with
the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal
cord, which contain full-length TDP-43, are frequently positive for this marker
protein. Amyotrophic lateral sclerosis (ALS) is a fatal human neurodegenerative disease
affecting primarily motor neurons. Two RNA-binding proteins, TDP-43 and FUS,
aggregate in the degenerating motor neurons of ALS patients, and mutations in
the genes encoding these proteins cause some forms of ALS. TDP-43 and FUS and
several related RNA-binding proteins harbor aggregation-promoting prion-like
domains that allow them to rapidly self-associate. This property is critical for
the formation and dynamics of cellular ribonucleoprotein granules, the crucibles
of RNA metabolism and homeostasis. Recent work connecting TDP-43 and FUS to
stress granules has suggested how this cellular pathway, which involves protein
aggregation as part of its normal function, might be coopted during disease
pathogenesis. Mutations of the FUS gene were first reported to cause amyotrophic lateral
sclerosis (ALS). Subsequent studies confirmed the role of mutations in ALS and
also implicated them in frontotemporal dementia (FTD). Recently, through
Next-Generation Exome sequencing approaches a mutation resulting in a
substitution (p.Q290X) in the nuclear export domain of the FUS protein was
nominated as a cause of autosomal domit essential tremor (ET) in a large
kindred. In addition, recent reports suggest a possible role for TDP-43
mutations in parkinsonism; TDP-43 is another RNA-binding protein implicated in
ALS. Given these findings we investigated the role of FUS variants in
Parkinson's disease (PD). We sequenced specific regions of the gene encoding
three functional domains of the FUS protein in 702 patients with PD. Our
sequencing study did not identify any novel non-synonymous variant that would
appear to affect the subjects' susceptibility to Parkinson's disease. These
findings and previous studies have shown that variants within the FUS gene are
not a common cause of PD or ET, in comparison to their role in ALS. Fused in sarcoma (FUS) is an RNA-binding protein involved in pathogenesis of
several neurodegenerative diseases. Aggregation of mislocalized FUS into
non-amyloid inclusions is believed to be pivotal in the development of cell
dysfunction, but the mechanism of their formation is unclear. Using transient
expression of a panel of deletion and chimeric FUS variants in various cultured
cells, we demonstrated that FUS accumulating in the cytoplasm nucleates a novel
type of RNA granules, FUS granules (FGs), that are structurally similar but not
identical to physiological RNA transport granules. Formation of FGs requires FUS
N-terminal prion-like domain and the ability to bind specific RNAs. Clustering
of FGs coupled with further recruitment of RNA and proteins produce larger
structures, FUS aggregates (FAs), that resemble but are clearly distinct from
stress granules. In conditions of attenuated transcription, FAs lose RNA and
dissociate into RNA-free FUS complexes that become precursors of large
aggresome-like structures. We propose a model of multistep FUS aggregation
involving RNA-dependent and RNA-independent stages. This model can be
extrapolated to formation of pathological inclusions in human FUSopathies. Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering
specific RNA-binding proteins, preventing them from performing their normal
functions. Conversely, mutations in RNA-binding proteins can trigger
neurodegeneration at least partly by altering RNA metabolism. In Fragile
X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR
of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients
and CGG repeats in isolation elicit toxicity in Drosophila and other animal
models. Here, we identify the amyotrophic lateral sclerosis (ALS)-associated
RNA-binding protein TAR DNA-binding protein (TDP-43) as a suppressor of CGG
repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appears
specific to TDP-43, as co-expression of another ALS-associated RNA-binding
protein, FUS, exacerbates the toxic effects of CGG repeats. Suppression of CGG
RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43
does not co-localize with CGG RNA foci and its ability to bind RNA is not
required for rescue. TDP-43-dependent rescue does, however, require fly hnRNP
A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of
TDP-43 that preclude interactions with hnRNP A2/B1 abolish TDP-43-dependent
rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity
by hnRNP A2/B1 is not affected by RNAi-mediated knockdown of the fly TDP-43
orthologue, TBPH. Lastly, TDP-43 suppresses CGG repeat-triggered mis-splicing of
an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43
suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and
suggest a convergence of pathogenic cascades between repeat expansion disorders
and RNA-binding proteins implicated in neurodegenerative disease. |
Is there a role for transcription factories in genome organization? | The mammalian nucleus is a highly complex structure that carries out a diverse range of functions such as DNA replication, cell division, RNA processing, and nuclear export/import. Many of these activities occur at discrete subcompartments that intersect with specific regions of the genome. Over the past few decades, evidence has accumulated to suggest that RNA transcription also occurs in specialized sites, called transcription factories, that may influence how the genome is organized. There may be certain efficiency benefits to cluster transcriptional activity in this way. However, the clustering of genes at transcription factories may have consequences for genome stability, and increase the susceptibility to recurrent chromosomal translocations that lead to cancer | When placed between an enhancer and promoter, certain DNA sequence elements
inhibit enhancer-stimulated gene expression. The best characterized of these
enhancer-blocking insulators, gypsy in Drosophila and the CTCF-binding element
in vertebrates and flies, stabilize contacts between distant genomic regulatory
sites leading to the formation of loop domains. Current results show that CTCF
mediates long-range contacts in the mouse beta-globin locus and at the
Igf2/H19-imprinted locus. Recently described active chromatin hubs and
transcription factories also involve long-range interactions; it is likely that
CTCF interferes with their formation when acting as an insulator. The properties
of CTCF, and its newly described genomic distribution, suggest that it may play
an important role in large-scale nuclear architecture, perhaps mediated by the
co-factors with which it interacts in vivo. Since the advent of FISH (fluorescence in situ hybridization), there have been
major advances in our understanding of how the genome is organized in interphase
nuclei. Indeed, this organization is found to be non-random and individual
chromosomes occupy discrete regions known as territories. Determining the
factors that drive the spatial positioning of these territories within nuclei
has caused much debate; however, in proliferating cells, there is evidently a
correlation between radial positioning and gene density. Indeed, gene-poor
chromosomes tend to be located towards the nuclear edge, while those that are
more gene-rich are positioned more internally. These observations pose a number
of questions: first, what is the function of this global organization and,
secondly, is it representative of that occurring at a more local scale? During
the course of this review, these questions will be considered, in light of the
current literature regarding the role of transcription factories and the nuclear
matrix in interphase genome organization. Molecular dynamics simulations are used to model proteins that diffuse to DNA,
bind, and dissociate; in the absence of any explicit interaction between
proteins, or between templates, binding spontaneously induces local DNA
compaction and protein aggregation. Small bivalent proteins form into rows [as
on binding of the bacterial histone-like nucleoid-structuring protein (H-NS)],
large proteins into quasi-spherical aggregates (as on oparticle binding), and
cylinders with eight binding sites (representing octameric nucleosomal cores)
into irregularly folded clusters (like those seen in nucleosomal strings).
Binding of RNA polymerase II and a transcription factor (NFκB) to the
appropriate sites on four human chromosomes generates protein clusters analogous
to transcription factories, multiscale loops, and intrachromosomal contacts that
mimic those found in vivo. We suggest that this emergent behavior of clustering
is driven by an entropic bridging-induced attraction that minimizes bending and
looping penalties in the template. The mammalian nucleus is a highly complex structure that carries out a diverse
range of functions such as DNA replication, cell division, RNA processing, and
nuclear export/import. Many of these activities occur at discrete
subcompartments that intersect with specific regions of the genome. Over the
past few decades, evidence has accumulated to suggest that RNA transcription
also occurs in specialized sites, called transcription factories, that may
influence how the genome is organized. There may be certain efficiency benefits
to cluster transcriptional activity in this way. However, the clustering of
genes at transcription factories may have consequences for genome stability, and
increase the susceptibility to recurrent chromosomal translocations that lead to
cancer. The relationships between genome organization, transcription, and
chromosomal translocation formation will have important implications in
understanding the causes of therapy-related cancers. |
Which are the Chompret criteria for Li-Fraumeni syndrome? | 1) According to the Chompret criteria for LFS, any patient with adrenocortical cancer (ACC), irrespective of age and family history, is at high risk for a TP53 germline mutation.
2) All families with a p53 mutation must have at least one family member with a sarcoma, breast, brain, or adrenocortical carcinoma (ACC). | PURPOSE: A clinical testing cohort was used to gain a broader understanding of
the spectrum of tumors associated with germline p53 mutations to aid clinicians
in identifying high-risk families.
PATIENTS AND METHODS: Full sequencing of the coding exons (2 to 11) and
associated splice junctions of the p53 gene was performed on 525 consecutive
patients whose blood samples were submitted for diagnostic testing. Clinical
features of p53 germline carriers in this cohort were characterized, clinical
referral schemes based on reported p53-associated family phenotypes were
evaluated, and practical mutation prevalence tables were generated.
RESULTS: Mutations were identified in 91 (17%) of 525 patients submitted for
testing. All families with a p53 mutation had at least one family member with a
sarcoma, breast, brain, or adrenocortical carcinoma (ACC). Every individual with
a choroid plexus tumor (eight of eight) and 14 of 21 individuals with a
childhood ACC had a mutation regardless of family history. Based on reported
personal and family history, 95% of patients (71 of 75) with a mutation met
either classic Li Fraumeni syndrome (LFS) or Chompret criteria. A simplified
prevalence table provides a concise summary of individual and family
characteristics associated with p53 mutations.
CONCLUSION: This is, to our knowledge, the largest single report of diagnostic
testing for germline p53 mutations, yielding practical mutation prevalence
tables and suggesting clinical utility of classic LFS and Chompret criteria for
identifying a subset of cancer-prone families with p53 germline mutations, with
important implications for diagnosis and management. CONTEXT: Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome
associated with germline mutations in TP53. According to the Chompret criteria
for LFS, any patient with adrenocortical cancer (ACC), irrespective of age and
family history, is at high risk for a TP53 germline mutation. However, whereas
such mutations have been detected with high frequency in childhood ACC, a large
cohort of adult patients with ACC has never been investigated for TP53 germline
mutations.
OBJECTIVE: The aim of the study was to evaluate the prevalence of TP53 germline
mutations in adult patients with ACC.
SUBJECTS AND METHODS: In 103 adult Caucasian patients with ACC, TP53 germline
mutation analysis was performed. In patients with a TP53 germline mutation,
tumor tissue was analyzed for loss of heterozygosity of TP53 and p53
immunohistochemistry. Family history and clinical course were also evaluated.
RESULTS: In four patients, a total of five TP53 germline mutations were found.
Two mutations occurred in exon 10 (R337H and I332M, respectively), outside the
hot spot region. Here, three mutations are described for the first time in ACC,
and one, which occurred combined with a second mutation (R202C) on the same
allele, has never been reported before in the context of LFS. This combined
mutation was associated with a remarkable family history of ACC also affecting
the mother and uncle of the index patient. In the 23 patients with ACC below the
age of 40 yr, 13% (95% confidence interval, 3.7-32.9%) carried a TP53 germline
mutation, whereas such mutations were rare in older patients with ACC.
CONCLUSION: Our findings indicate a need to revise the Chompret criteria.
However, in younger adults (<40 yr old) with ACC, screening for TP53 germline
mutations may be justified. |
When ceritinib used instead of crizotinib? | Ceritinib is approved for the treatment of ALK-positive metastatic NSCLC patients that are crizotinib-resistant and crizotinib-naïve. | BACKGROUND: Non-small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma
kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib,
but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor
that has shown greater antitumor potency than crizotinib in preclinical studies.
METHODS: In this phase 1 study, we administered oral ceritinib in doses of 50 to
750 mg once daily to patients with advanced cancers harboring genetic
alterations in ALK. In an expansion phase of the study, patients received the
maximum tolerated dose. Patients were assessed to determine the safety,
pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies
were performed before ceritinib treatment to identify resistance mutations in
ALK in a group of patients with NSCLC who had had disease progression during
treatment with crizotinib.
RESULTS: A total of 59 patients were enrolled in the dose-escalation phase. The
maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic
events included diarrhea, vomiting, dehydration, elevated aminotransferase
levels, and hypophosphatemia. This phase was followed by an expansion phase, in
which an additional 71 patients were treated, for a total of 130 patients
overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib
per day, the overall response rate was 58% (95% confidence interval [CI], 48 to
67). Among 80 patients who had received crizotinib previously, the response rate
was 56% (95% CI, 45 to 67). Responses were observed in patients with various
resistance mutations in ALK and in patients without detectable mutations. Among
patients with NSCLC who received at least 400 mg of ceritinib per day, the
median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5).
CONCLUSIONS: Ceritinib was highly active in patients with advanced,
ALK-rearranged NSCLC, including those who had had disease progression during
crizotinib treatment, regardless of the presence of resistance mutations in ALK.
(Funded by Novartis Pharmaceuticals and others; ClinicalTrials.gov number,
NCT01283516.). Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK)
gene rearrangements invariably develop resistance to the ALK tyrosine kinase
inhibitor (TKI) crizotinib. Herein, we report the first preclinical evaluation
of the next-generation ALK TKI, ceritinib (LDK378), in the setting of crizotinib
resistance. An interrogation of in vitro and in vivo models of acquired
resistance to crizotinib, including cell lines established from biopsies of
patients with crizotinib-resistant NSCLC, revealed that ceritinib potently
overcomes crizotinib-resistant mutations. In particular, ceritinib effectively
inhibits ALK harboring L1196M, G1269A, I1171T, and S1206Y mutations, and a
cocrystal structure of ceritinib bound to ALK provides structural bases for this
increased potency. However, we observed that ceritinib did not overcome two
crizotinib-resistant ALK mutations, G1202R and F1174C, and one of these
mutations was identified in 5 of 11 biopsies from patients with acquired
resistance to ceritinib. Altogether, our results demonstrate that ceritinib can
overcome crizotinib resistance, consistent with clinical data showing marked
efficacy of ceritinib in patients with crizotinib-resistant disease.
SIGNIFICANCE: The second-generation ALK inhibitor ceritinib can overcome several
crizotinib-resistant mutations and is potent against several in vitro and in
vivo laboratory models of acquired resistance to crizotinib. These findings
provide the molecular basis for the marked clinical activity of ceritinib in
patients with ALK-positive NSCLC with crizotinib-resistant disease. Cancer
Discov; 4(6); 662-73. ©2014 AACR. See related commentary by Ramalingam and
Khuri, p. 634 This article is highlighted in the In This Issue feature, p. 621. The anaplastic lymphoma kinase (ALK) gene plays a key role in the pathogenesis
of selected tumors, including non-small-cell lung cancer (NSCLC). Patients with
ALK-rearranged NSCLC are initially sensitive to the ALK inhibitor crizotinib but
eventually become resistant, limiting its therapeutic potential. Ceritinib is an
oral second-generation ALK inhibitor with greater preclinical antitumor potency
than crizotinib in ALK-positive NSCLC. A Phase I trial of ceritinib in
ALK-positive tumors demonstrated good activity in patients with advanced NSCLC,
including those who had progressed on crizotinib. Adverse events are similar to
those seen with other ALK tyrosine kinase inhibitors and are generally
manageable. Ongoing trials are evaluating ceritinib in patients with
ALK-rearranged NSCLC treated with prior chemotherapy and/or crizotinib. Non-small cell lung cancer (NSCLC) represents the paradigm of personalized
treatment in human cancer. Several oncogenic alterations with druggable
potential have been identified, with ALK rearrangements representing one of the
newest and most appealing. Crizotinib is now recognized as the standard of care
in chemotherapy-pretreated ALK-positive NSCLC due to the positive results of a
recently published trial. Unfortunately, no patient exposed to crizotinib can be
cured, and after a median time of 1 year, resistance inevitably occurs.
Overcoming resistance is the major challenge in clinical oncology and many
molecules are currently under evaluation, including ceritinib (LDK-378).
Ceritinib is an oral, potent, second-generation ALK inhibitor recently approved
by the U.S. Food and Drug Administration. Preclinical data showed impressive
antitumor activity against crizotinib-resistant clones, and based on available
data, ceritinib could represent a suitable option in crizotinib-resistant NSCLC. The success in identifying the chromosomal rearrangements involving the
anaplastic lymphoma kinase (ALK) as an oncogenic driver has thoroughly changed
the treatment of non-small-cell lung cancer. In the past decade, targeted drugs
have emerged as an efficient personalized strategy for ALK-rearranged
non-small-cell lung cancer. The accelerated approval of potent ALK inhibitors,
such as crizotinib and more recently ceritinib (LDK378), based on the well
designed phase I/II trials has been a landmark success in clinical cancer
research and contributes a new era of oncogenic targeted therapy characterized
by elegant clinical trial design. In this review, we aim to present the current
knowledge on acquired resistance of crizotinib known as a first-in-class ALK
inhibitor and potential solutions to improve the cost-effectiveness, and to
review the difference between ceritinib and crizotinib; preclinical data and
results of the elegant early clinical trial of ceritinib which promoted its
accelerated approval, pharmacokinetics, safety profile, and tolerability, the
updated results (eg, efficacy on brain metastases), and robust design of ongoing
phase II/III trials, and future directions of ceritinib to be a potent
alternative to crizotinib for ALK-rearranged non-small-cell lung cancer are also
presented. In recent years, many personalized treatments have been developed for NSCLC
(non-small-cell lung cancer) patients. Among these, gefitinib, erlotinib, and
afatinib are selective epidermal growth factor receptor (EGFR) tyrosine kinase
inhibitors for patients with EGFR gene mutations, while crizotinib and ceritinib
are two new tyrosine kinase inhibitors directed against the echinoderm
microtubule-like protein 4-anaplastic lymphoma kinase translocation. The
possibility of these new molecules being used to treat patients without
adenocarcinoma histology is notably small. For example, EGFR mutations and
anaplastic lymphoma kinase fusion gene rearrangement are rare in patients with
squamous cell carcinoma (generally <1%). Additionally, the benefit of targeted
treatment approaches in patients with small-cell lung cancer histology is
limited. All of these findings highlight the distinctive nature of
adenocarcinoma of the lung among all lung cancer subtypes. Unfortunately, to
date, less than 15% of patients with adenocarcinoma of the lung are ideal
candidates for these targeted therapies. |
What type of enzyme is peroxiredoxin 2 (PRDX2)? | Peroxiredoxin 2 (PRDX2) is an antioxidant enzyme that uses cysteine residues to decompose peroxides.
Peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides
Peroxiredoxin 2 (Prx2) is a thiol-dependent peroxidase. | Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant
protein in the erythrocyte, and its absence in knock-out mice gives rise to
hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely
sensitive to oxidation by H(2)O(2), as dimerization was observed after exposure
of 5 x 10(6) cells/mL to 0.5 muM H(2)O(2). In contrast to Prx2 in Jurkat T
lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine
thiol to a sulfinic/sulfonic acid) in erythrocytes. Reduction of dimerized Prx2
in the erythrocyte occurred very slowly, with reversal occurring gradually over
a 20-minute period. Very low thioredoxin reductase activity was detected in
hemolysates. We postulate that this limits the rate of Prx2 regeneration, and
this inefficiency in recycling prevents the overoxidation of Prx2. We also found
that Prx2 was oxidized by endogenously generated H(2)O(2), which was mainly
derived from hemoglobin autoxidation. Our results demonstrate that in the
erythrocyte Prx2 is extremely efficient at scavenging H(2)O(2) noncatalytically.
Although it does not act as a classical antioxidant enzyme, its high
concentration and substrate sensitivity enable it to handle low H(2)O(2)
concentrations efficiently. These unique redox properties may account for its
nonredundant role in erythrocyte defense against oxidative stress. We investigated high- or low-dose irradiation-responsive proteins using
proteomics on two-dimensional (2D) PAGE, and the effects of ageing on cell
responses to radiation in variously aged rat astrocytes. After 5 Gy irradiation,
the relative abundance of peroxiredoxin 2, an antioxidant enzyme, and latexin,
an inhibitor of carboxypeptidase, increased. The induction of these proteins was
suppressed by ageing, suggesting that the response to high-dose radiation
decreased with ageing. The relative abundance of elongation factor 2 (EF-2)
fragment increased 3 h and reduced 24 h after 0.1 Gy irradiation. Temporal
enhancement of the EF-2 fragment due to low-dose irradiation was suppressed by
ageing. Since radiation adaptive response in cultured astrocytes was observed 3
h but not 24 h after 0.1 Gy irradiation and suppressed by ageing as previously
reported, alteration of the EF-2 fragment corresponded to the radiation adaptive
response. We also examined phospho-protein profiles, resulting in the relative
abundance of phospho-EF-1beta and phospho-beta-actin being altered by 0.1 Gy
irradiation; however, ageing did not affect the alteration of phospho-EF-1beta
and phospho-beta-actin, unlike the EF-2 fragment. The results suggested that the
EF-2 fragment was a possible candidate for the protein responsible for the
radiation adaptive response in cultured astrocytes. The mechanisms underlying lithium's therapeutic efficacy in the chronic
treatment of bipolar disorder are not clearly understood. Useful insights can be
obtained by identifying genes that are differentially regulated during chronic
lithium treatment. Toward this end, we have used microarray technology to
identify mRNAs that are differentially expressed in a human neuronal cell line
that has been continuously maintained in therapeutic levels of lithium for 33
days. Significantly, unlike other transcriptomes where predomitly rodent
cells were used and a limited number of genes probed, we have used human cells
probed with more extensive 44,000 gene microarrays. A total of 671
differentially regulated transcripts, after correcting for false discovery
rates, were identified, of which 347 and 324, respectively, were found to be up-
and downregulated. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, was the most
upregulated while tribbles homolog 3 (TRB3), a pro-apoptotic protein, was the
most downregulated, implying a beneficial effect of lithium on neuronal cells.
Several of the most highly regulated genes are novel, uncharacterized and encode
proteins of unknown function. Differentially expressed genes associated with
phosphoinositide metabolism include those encoding phosphatidyl inositol
4-phosphate 5-kinase type II alpha (PIP5K2A), WD repeat domain, phosphoinositide
interacting 1 protein (WIPI49), tribbles homolog 3 (TRB3) and sorting nexin 14
(SNX14). A protein interactome using some of the saliently regulated genes
identified protein kinase C (PKC) as a major target for lithium action while a
global analysis of all 671 differentially expressed genes identified the
mitogen-activated protein kinase pathway as the most regulated. The list of
highly regulated genes, besides encoding putative targets for antimanic agents,
should prove useful in defining novel pathways, or to better understand the
mechanisms, underlying the mood stabilization process. Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5
(cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by
which cdk5 promotes death is unclear. Complicating the role of cdk5 are the
observations that cdk5 can exist in multiple cellular regions and possess both
prosurvival and prodeath characteristics. In particular, the critical role of
cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we
determined where cdk5 was activated in models of ischemia and how manipulation
of cdk5 in differing compartments may affect neuronal death. Here, we show a
critical function for cytoplasmic cdk5 in both focal and global models of
stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5
localized to the cytoplasm is protective. Importantly, we also demonstrate the
antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of
cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent.
Following focal ischemia, nuclear cdk5 is activated and functionally relevant
while there is no evidence for such activation following global ischemia.
Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear
target of cdk5 in vitro, is also phosphorylated by cdk5 following focal
ischemia. In addition, MEF2D expression in this paradigm ameliorates death.
Together, our results address the critical issue of cdk5 activity
compartmentalization, as well as define critical substrates for both cytoplasmic
and nuclear cdk5 activity in adult models of stroke. Peroxiredoxin-2 (Prdx2), a potent peroxide reductant, is the third most abundant
protein in the erythrocyte and might be expected to play a major role in the
cell's oxidative defenses. However, in this study, experiments with erythrocytes
from mice with a disrupted Prdx2 gene found that the cells were not more
sensitive to exogenous H(2)O(2) or organic peroxides than wild type.
Intraerythrocytic H(2)O(2) was increased, however, indicating an important role
for Prdx2 in detoxifying endogenously generated H(2)O(2). These results are
consistent with proposals that red cell Prdx2 acts stoichiometrically, not
catalytically, in reducing peroxides. Additional experiments with mice with
disrupted catalase or glutathione peroxidase (Gpx1) genes showed that Gpx1 is
the only erythrocyte enzyme that reduces organic peroxides. Catalase(-/-) cells
were readily oxidized by exogenous H(2)O(2). Cells lacking both catalase and
Gpx1 were more sensitive to exogenous H(2)O(2) than cells lacking only catalase.
A kinetic model proposed earlier to rationalize results with Gpx1(-/-)
erythrocytes also fits the data with Prdx2(-/-) cells and indicates that
although Gpx1 and Prdx2 both participate in removing endogenous H(2)O(2), Prdx2
plays a larger role. Although the rate of H(2)O(2) production in the red cell is
quite low, Prdx2-deficient mice are anemic, suggesting an important role in
erythropoiesis. Anti-endothelial cell antibodies (AECA) have been frequently detected in
systemic vasculitis, which affects blood vessels of various sizes. To understand
the pathogenic roles of AECA in systemic vasculitis, we attempted to identify
target antigens for AECA comprehensively by a proteomic approach. Proteins
extracted from human umbilical vein endothelial cells (HUVEC) were separated by
two-dimensional electrophoresis, and Western blotting was subsequently conducted
using sera from patients with systemic vasculitis. As a result, 53 autoantigenic
protein spots for AECA were detected, nine of which were identified by mass
spectrometry. One of the identified proteins was peroxiredoxin 2 (Prx2), an
anti-oxidant enzyme. Frequency of anti-Prx2 autoantibodies, measured by
enzyme-linked immunosorbent assay (ELISA), was significantly higher in systemic
vasculitis (60%) compared to those in collagen diseases without clinical
vasculitis (7%, P < 0·01) and healthy individuals (0%, P < 0·01). Further, the
titres changed in parallel with the disease activity during time-courses. The
presence of anti-Prx2 autoantibodies correlated significantly with elevation of
serum d-dimers and thrombin-antithrombin complex (P < 0·05). Immunocytochemical
analysis revealed that live endothelial cells expressed Prx2 on their surface.
Interestingly, stimulation of HUVEC with rabbit anti-Prx2 antibodies increased
secretion of interleukin (IL)-6, IL-1β, IL-1ra, growth regulated oncogene
(GRO)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage
colony-stimulating factor (GM-CSF), IL-8 and monocyte chemoattractant protein
(MCP)-1 more than twofold compared to that of with rabbit immunoglobulin (Ig)G.
Taken together, our data suggest that anti-Prx2 autoantibodies would be a useful
marker for systemic vasculitis and would be involved in the inflammatory
processes of systemic vasculitis. Peroxiredoxin 2 (PRDX2) has been known to act as an antioxidant enzyme whose
main function is H(2)O(2) reduction in cells. We aimed to study the expression
patterns of PRDX2 in mouse ovaries and explore the function of this protein in
apoptosis of granulosa cells (GCs). We found that the expression of the PRDX2
protein in atretic follicle GCs was markedly higher than in healthy follicle
GCs. In vitro, the transfection of siRNA targeting the Prdx2 gene inhibited the
proliferation and induced the apoptosis of primary cultured GCs. Furthermore,
suppression of PRDX2 resulted in the augmentation of endogenous H(2)O(2), and
the ability to eliminate the exogenous H(2)O(2) was attenuated. The expression
of PRDX2 and nuclear factor kappa-light-chain-enhancer of activated B cells
(NFKB), whose activity was inhibited by binding to IKB, increased in GCs treated
with various concentrations of H(2)O(2) for 30 min. However, no significant
change in cytoplasmic IKB expression was observed. At 2 h after treatment with
H(2)O(2), nuclear NFKB expression level was reduced, cytoplasmic IKB expression
was increased, and PRDX2 expression was unchanged. Silencing of the Prdx2 gene
caused early changes in NFKB and IKB expression in the primary cultured GCs
compared to that in control cells. Taken together, these data suggest that PRDX2
plays an important role in inhibiting apoptosis in GCs and that PRDX2 actions
may be related to the expression of NFKB and IKB. AIM: Many studies have reported that the generation of reactive oxygen species
(ROS) increases during the differentiation of muscle-derived C2C12 cells.
Peroxiredoxin-2 (Prx-2) is an abundant mammalian enzyme that protects against
oxidative stress. However, the role of Prx-2 in muscle differentiation has not
been investigated.
RESULTS: In this study, we demonstrated that Prx-2 expression increases during
muscle differentiation and regeneration in response to exogenous H(2)O(2). This
increase occurs only in myoblast cell lines because no increase in Prx-2
expression was observed in the NIH3T3, MEF, Chang, or HEK293 cell lines. The
antioxidants, N-acetyl L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzenedisulfonic
acid (Tiron), both suppressed myogenesis and Prx-2 expression. Moreover, Prx-2
was upregulated at the transcriptional level by NF-κB during the differentiation
of muscle-derived C2C12 cells. We also found that inhibition of
phosphatidylinositol 3-kinase (PI3K) blocks NF-κB activation and suppresses
Prx-2 expression. Interestingly, Prx-2 knockdown increased the expression levels
of other antioxidant enzymes, including all of the other Prx family member,
thioredoxin-1 (Trx-1) and catalase, but also enhanced the accumulation of
endogenous ROS during muscle differentiation.
INNOVATION: In this study, we demonstrated for the first time that Prx-2 is
unregulated during the muscle differentiation and regeneration.
CONCLUSION: Prx-2 is upregulated via the PI3K/NF-κB pathway and attenuates
oxidative stress during muscle differentiation and regeneration. Reactive oxygen species are involved in ovulation. The aim of this study was to
examine gonadotropin regulation of antioxidant enzyme sulfiredoxin (Srx) and
peroxiredoxin 2 (PRDX2) expressions and modification during the ovulatory
process in rats. Administration of antioxidants in vivo reduced ovulation rate
and cumulus expansion. LH treatment increased H(2)O(2) levels within 15 min,
which, in turn, induced Srx gene expression in cultured preovulatory follicles.
Treatment of preovulatory follicles with catalase suppressed the stimulatory
effect of LH on Akt phosphorylation. LH- or H(2)O(2)-stimulated Srx mRNA levels
were suppressed by inhibitors of antioxidant agents and MAPK kinase. An in vivo
injection of equine chorionic gonadotropin-human chorionic gonadotropin (hCG)
stimulated Srx mRNA within 1 h in granulosa but not thecal cells of preovulatory
follicles. Srx protein levels were stimulated from 3 h post-hCG injection.
Immunofluorescence analysis revealed that oocytes expressed the Srx protein.
Furthermore, hCG treatment increased Srx expression in mural granulosa, theca
and cumulus cells, but the Srx protein was not detected in corpora lutea. Gene
expression of PRDX2, identified as an Srx-dependent modified enzyme, was
stimulated by gonadotropins. In situ hybridization analysis demonstrated that
PRDX2 mRNA was detected in oocytes and theca cells as well as granulosa cells of
some antral and preovulatory follicles. High levels of PRDX2 mRNA were detected
in corpora lutea. Total levels of PRDX2 protein were not changed by
gonadotropins. However, levels of hyperoxidized PRDX2 increased within 2-3 h
after the hCG injection. Taken together, gonadotropin stimulation of Srx
expression and PRDX2 modification in the ovary suggest the existence of an
antioxidant system to maintain H(2)O(2) production and elimination during the
periovulatory period. In melanoma, transition to the vertical growth phase is the critical step in
conversion to a deadly maligt disease. Here, we offer the first evidence that
an antioxidant enzyme has a key role in this transition. We found that the
antioxidant enzyme peroxiredoxin-2 (Prx2) inversely correlated with the
metastatic capacity of human melanoma cells. Silencing Prx2 expression
stimulated proliferation and migration, whereas ectopic expression of Prx2
produced the opposite effect. Mechanistic investigations indicated that Prx2
negatively regulated Src/ERK activation status, which in turn fortified adherens
junctions function by increasing E-cadherin expression and
phospho-Y654-dependent retention of β-catenin in the plasma membrane. In murine
melanoma cells, Prx2 silencing enhanced lung metastasis in vivo. Interestingly,
the natural compound gliotoxin, which is known to exert a Prx-like activity,
inhibited proliferation and migration as well as lung metastasis of
Prx2-deficient melanoma cells. Overall, our findings reveal that Prx2 is a key
regulator of invasion and metastasis in melanoma, and also suggest a
pharmacologic strategy to effectively decrease deadly maligt forms of this
disease. PURPOSE: Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein
critical for cell function. This study examined whether the levels of lymphocyte
PRDX-2 are altered over 1 month following ultra-endurance exercise.
METHODS: Nine middle-aged men undertook a single-stage, multi-day 233 km (145
mile) ultra-endurance running race. Blood was collected immediately before
(Pre), upon completion/retirement (Post), and following the race at Day 1, Day 7
and Day 28. Lymphocyte lysates were examined for PRDX-2 by reducing and
non-reducing SDS-PAGE with western blotting. In a sub-group of men who completed
the race (n = 4), PRDX-2 oligomeric state (indicative of redox status) was
investigated.
RESULTS: Ultra-endurance exercise caused significant changes in lymphocyte
PRDX-2 (F(4,32) 3.409, p = 0.020, η(2) = 0.299): 7 days after the race, PRDX-2
levels in lymphocytes had fallen to 30% of pre-race values (p = 0.013) and
returned to near-normal levels at Day 28. Non-reducing gels demonstrated that
dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in three of
four race completers immediately post-race, indicative of an 'antioxidant
response'. Moreover, monomeric PRDX-2 was also increased immediately post-race
in two of four race-completing subjects, indicative of oxidative damage, which
was not detectable by Day 7.
CONCLUSIONS: Lymphocyte PRDX-2 was decreased below normal levels 7 days after
ultra-endurance exercise. Excessive accumulation of reactive oxygen species
induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2
by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2
could influence cell function and might, in part, explain reports of
dysregulated immunity following ultra-endurance exercise. |
Is the length of the poly(A) tail involved in human disease? | Yes. Severely truncated poly(A) tails of mitochondrial mRNAs were found to be involved in an autosomal recessive spastic ataxia with optic atrophy. | |
Why graphics processing units (GPU) are more suitable for biological tasks than central processing units (CPU)? | Traditional central processing unist (CPUs) are reaching their limit in processing power and are not designed primarily for multithreaded applications. Graphics processing units (GPUs) on the other hand are affordable, scalable computer powerhouses that, thanks to the ever increasing demand for higher quality graphics, have yet to reach their limit. Typically high-end CPUs have 8-16 cores, whereas GPUs can have more than 2,500 cores. GPUs are also, by design, highly parallel, multicore and multithreaded, able of handling thousands of threads doing the same calculation on different subsets of a large data set. This ability is what makes them perfectly suited for biological analysis tasks. Lately this potential has been realized by many bioinformatics researches and a huge variety of tools and algorithms have been ported to GPUs, or designed from the ground up to maximize the usage
of available cores. | There is currently a strong push in the research community to develop biological
scale implementations of neuron based vision models. Systems at this scale are
computationally demanding and generally utilize more accurate neuron models,
such as the Izhikevich and the Hodgkin-Huxley models, in favor of the more
popular integrate and fire model. We examine the feasibility of using graphics
processing units (GPUs) to accelerate a spiking neural network based character
recognition network to enable such large scale systems. Two versions of the
network utilizing the Izhikevich and Hodgkin-Huxley models are implemented.
Three NVIDIA general-purpose (GP) GPU platforms are examined, including the
GeForce 9800 GX2, the Tesla C1060, and the Tesla S1070. Our results show that
the GPGPUs can provide significant speedup over conventional processors. In
particular, the fastest GPGPU utilized, the Tesla S1070, provided a speedup of
5.6 and 84.4 over highly optimized implementations on the fastest central
processing unit (CPU) tested, a quadcore 2.67 GHz Xeon processor, for the
Izhikevich and the Hodgkin-Huxley models, respectively. The CPU implementation
utilized all four cores and the vector data parallelism offered by the
processor. The results indicate that GPUs are well suited for this application
domain. We investigate the potential in using of using a graphics processor unit (GPU)
for Monte-Carlo (MC)-based radiation dose calculations. The percent depth dose
(PDD) of photons in a medium with known absorption and scattering coefficients
is computed using a MC simulation running on both a standard CPU and a GPU. We
demonstrate that the GPU's capability for massive parallel processing provides a
significant acceleration in the MC calculation, and offers a significant
advantage for distributed stochastic simulations on a single computer.
Harnessing this potential of GPUs will help in the early adoption of MC for
routine planning in a clinical environment. Theoretical exploration of fundamental biological processes involving the forced
unraveling of multimeric proteins, the sliding motion in protein fibers and the
mechanical deformation of biomolecular assemblies under physiological force
loads is challenging even for distributed computing systems. Using a C(α)-based
coarse-grained self organized polymer (SOP) model, we implemented the Langevin
simulations of proteins on graphics processing units (SOP-GPU program). We
assessed the computational performance of an end-to-end application of the
program, where all the steps of the algorithm are running on a GPU, by profiling
the simulation time and memory usage for a number of test systems. The ∼90-fold
computational speedup on a GPU, compared with an optimized central processing
unit program, enabled us to follow the dynamics in the centisecond timescale,
and to obtain the force-extension profiles using experimental pulling speeds
(v(f) = 1-10 μm/s) employed in atomic force microscopy and in optical
tweezers-based dynamic force spectroscopy. We found that the mechanical
molecular response critically depends on the conditions of force application and
that the kinetics and pathways for unfolding change drastically even upon a
modest 10-fold increase in v(f). This implies that, to resolve accurately the
free energy landscape and to relate the results of single-molecule experiments
in vitro and in silico, molecular simulations should be carried out under the
experimentally relevant force loads. This can be accomplished in reasonable
wall-clock time for biomolecules of size as large as 10(5) residues using the
SOP-GPU package. BACKGROUND: Protein-DNA docking is a very challenging problem in structural
bioinformatics and has important implications in a number of applications, such
as structure-based prediction of transcription factor binding sites and rational
drug design. Protein-DNA docking is very computational demanding due to the high
cost of energy calculation and the statistical nature of conformational sampling
algorithms. More importantly, experiments show that the docking quality depends
on the coverage of the conformational sampling space. It is therefore desirable
to accelerate the computation of the docking algorithm, not only to reduce
computing time, but also to improve docking quality.
METHODS: In an attempt to accelerate the sampling process and to improve the
docking performance, we developed a graphics processing unit (GPU)-based
protein-DNA docking algorithm. The algorithm employs a potential-based energy
function to describe the binding affinity of a protein-DNA pair, and integrates
Monte-Carlo simulation and a simulated annealing method to search through the
conformational space. Algorithmic techniques were developed to improve the
computation efficiency and scalability on GPU-based high performance computing
systems.
RESULTS: The effectiveness of our approach is tested on a non-redundant set of
75 TF-DNA complexes and a newly developed TF-DNA docking benchmark. We
demonstrated that the GPU-based docking algorithm can significantly accelerate
the simulation process and thereby improving the chance of finding near-native
TF-DNA complex structures. This study also suggests that further improvement in
protein-DNA docking research would require efforts from two integral aspects:
improvement in computation efficiency and energy function design.
CONCLUSIONS: We present a high performance computing approach for improving the
prediction accuracy of protein-DNA docking. The GPU-based docking algorithm
accelerates the search of the conformational space and thus increases the chance
of finding more near-native structures. To the best of our knowledge, this is
the first ad hoc effort of applying GPU or GPU clusters to the protein-DNA
docking problem. To evaluate the use of general-purpose graphics processing units (GPGPUs) to
improve the performance of MODFLOW, an unstructured preconditioned conjugate
gradient (UPCG) solver has been developed. The UPCG solver uses a compressed
sparse row storage scheme and includes Jacobi, zero fill-in incomplete, and
modified-incomplete lower-upper (LU) factorization, and generalized
least-squares polynomial preconditioners. The UPCG solver also includes options
for sequential and parallel solution on the central processing unit (CPU) using
OpenMP. For simulations utilizing the GPGPU, all basic linear algebra operations
are performed on the GPGPU; memory copies between the central processing unit
CPU and GPCPU occur prior to the first iteration of the UPCG solver and after
satisfying head and flow criteria or exceeding a maximum number of iterations.
The efficiency of the UPCG solver for GPGPU and CPU solutions is benchmarked
using simulations of a synthetic, heterogeneous unconfined aquifer with tens of
thousands to millions of active grid cells. Testing indicates GPGPU speedups on
the order of 2 to 8, relative to the standard MODFLOW preconditioned conjugate
gradient (PCG) solver, can be achieved when (1) memory copies between the CPU
and GPGPU are optimized, (2) the percentage of time performing memory copies
between the CPU and GPGPU is small relative to the calculation time, (3)
high-performance GPGPU cards are utilized, and (4) CPU-GPGPU combinations are
used to execute sequential operations that are difficult to parallelize.
Furthermore, UPCG solver testing indicates GPGPU speedups exceed parallel CPU
speedups achieved using OpenMP on multicore CPUs for preconditioners that can be
easily parallelized. An efficient graphics processing units (GPUs) version of time-dependent
wavepacket code is developed for the atom-diatom state-to-state reactive
scattering processes. The propagation of the wavepacket is entirely calculated
on GPUs employing the split-operator method after preparation of the initial
wavepacket on the central processing unit (CPU). An additional split-operator
method is introduced in the rotational part of the Hamiltonian to decrease
communication of GPUs without losing accuracy of state-to-state information. The
code is tested to calculate the differential cross sections of H + H2 reaction
and state-resolved reaction probabilities of nonadiabatic triplet-singlet
transitions of O((3)P,(1)D) + H2 for the total angular momentum J = 0. The
global speedups of 22.11, 38.80, and 44.80 are found comparing the parallel
computation of one GPU, two GPUs by exact rotational operator, and two GPU
versions by an approximate rotational operator with serial computation of the
CPU, respectively. |
Which are the thyroid hormone analogs utilized in human studies? | TRIAC and TETRAC are two different thyroid hormone analogs utilized in human studies | A protein that binds tetraiodothyroacetic acid (tetrac) with high specificity
has been detected in normal rabbit serum. Scatchard plots revealed the protein
to have a principal binding site with both high capacity and high affinity for
tetrac (KA 4.8 X 10(10) M-1. Binding of tetrac by the protein is partially
inhibited by barbital. During polyacrylamide gel electrophoresis at pH 8.0, the
tetrac binding protein has a mobility characteristic of a prealbumin. As judged
from competitive binding studies, the protein also binds tetraiodothyropropionic
acid (tetraprop) firmly, but less so than tetrac. The apparent affinity of the
protein for the triiodinated analogues of tetrac and tetraprop is only about 1%
that for tetrac, and that for thyroxine (T4) only 0.6% that for tetrac;
3,5,3'-triiodothyronine is not bound at all. The protein can be utilized in a
competitive protein binding assay for tetrac in human serum, after removal of
cross-reacting T4. Thyroid hormones influence the expression of transmitter-specific enzymes by
central cholinergic neurons. Based on the fact that these cholinergic neurons
degenerate selectively in human Alzheimer's disease, it was hypothesized that
thyroid hormones might be beneficial in its treatment. However, since thyroid
hormones influence the function of most peripheral organs, derivatives selective
for central cholinergic neurons are necessary. The structural requirements of
the receptor mediating the effects of the thyroid hormones on central
cholinergic neurons were therefore compared with those of the receptors
mediating actions on peripheral organs. Cultures were prepared of dissociated
neurons from the septal area of fetal rat brains, and the differentiation of
cholinergic neurons was assessed by measuring the activity of choline
acetyltransferase (ChAT). Triiodothyronine (T3) was found to stimulate ChAT
activity in a dose-dependent manner. The effect of T3 was additive to that of
nerve growth factor. The potency of derivatives of T3 in elevating ChAT activity
in the cultures was compared with their known anti-goiter activity determined in
vivo and their binding affinity to the hepatic nuclear receptor measured in
vitro. The findings indicate that the structural requirements of central and
peripheral receptors are similar and that it therefore appears unlikely that
analogs of thyroid hormones can be developed which selectively affect
cholinergic neurons. 3,5,3,'-Triiodothyroacetic acid (Triac) has been used in therapy of resistance
to thyroid hormone on an empirical basis and appears beneficial in some studies.
We observed that the T3 analogs, Triac and 3,5,3'-triiodothyropropionic acid
(Triprop), have a higher affinity for the thyroid hormone receptor-beta 1 (TR
beta 1) than does T3 (2.7- and 1.8-fold, respectively), whereas the affinities
of the three compounds for TR alpha 1 are the same. To evaluate whether T3
analogs would have a differential effect on TR beta 1 and TR beta 1 mutants and
thus be a specific treatment for patients with resistance to thyroid hormone, we
examined the induction of the transcriptional activation of wild-type (wt) TR
alpha 1, TR beta 1, and mutant TR beta 1s by T3, Triac, and Triprop. The dose
response of transcriptional activation by T3 analogs was measured by transient
cotransfections with TRs and a rat malic enzyme-TRE fused to thymidine kinase
(TK)-chloramphenicol acetyltransferase (CAT) in COS-1 cells. For TR alpha 1 wt,
induction of CAT activity by T3 and Triac occurred at the same concentration.
For TR beta 1 wt, Triac and Triprop showed a higher maximal activity than T3
(Tripro > Triac > T3) and reached 50% induction at a lower concentration than T3
(Tripro < Triac < T3). Induction of CAT activity in five mutant TR beta 1s
(kindreds Mh, Mc, CL, Mf, and GH) was also analyzed. Even high levels of T3
analogs could not restore CAT activity to that of TR beta 1wt for any mutant. A
domit negative effect was produced by Mh, Mc, and Mf. Mutants CL and GH had a
mild domit negative effect depending on T3 analog concentrations and TREs.
Cotransfection studies were performed using a rat malic enzyme-TK-CAT reporter
plasmid to analyze the effects of hormones at near-physiological concentrations
of T3 and Triac. Triac had a significantly higher transcriptional activation
than T3 in Mc, CL, and GH, suggesting that Triac would have a beneficial effect
to different degrees for different mutant TR beta 1s. Using mutants Mc and GH,
further studies were carried out using rat GH and double palindromic and
inverted palindromic TREs in COS-1 cells. On each TRE, 10 nmol/L Triac induced
higher transcriptional activation in TR beta 1wt, mutant TR beta 1s, and TR beta
1wt plus mutant TR beta 1s (1:1 ratio) than the same dose of T3.(ABSTRACT
TRUNCATED AT 400 WORDS) This study investigated the response of TSH secretion to 3,5,3'-triiodothyronine
(T3) and 3,5,3'-triiodothyroacetic acid (Triac) in patients with resistance to
thyroid hormone, and compared the responses with those in patients with
TSH-secreting pituitary adenoma and normal subjects. A short-term administration
of 75 microg of T3 daily for 7 days suppressed serum TSH concentrations almost
completely in normal subjects, but suppressed TSH only partially in patients
with resistance to thyroid hormone and TSH-secreting pituitary adenoma. A
single-dose administration of 75 microg of T3 gave similar results in regard to
TSH suppressibility in these three subjects groups. In contrast, a single-dose
administration of 1.4 mg of Triac remarkably suppressed serum TSH concentrations
after 2 hours in not only normal subjects (-34 +/- 11% [mean +/- SD] from the
basal value) but also in patients with resistance to thyroid hormone (-31 +/-
9%), and this TSH suppression continued for 4 hours. After 24 hours, this TSH
suppression persisted in normal subjects (-62 +/- 12%) but was relieved in
patients with resistance to thyroid hormone (-23 +/- 14%). After the Triac
administration, molar ratios of alpha-subunit to TSH in serum were decreased in
patients with TSH-secreting pituitary adenoma but increased in patients with
resistance to thyroid hormone. Because the Triac therapy for patients with
resistance to thyroid hormone suppressed pituitary-TSH secretion during the
early phase of drug ingestion, this drug should be given several times within a
day to obtain continuous TSH-suppressive effects. We have recently described the proangiogenesis effects of thyroid hormone in the
chick chorioallantoic membrane (CAM) model. Generation of new blood vessels from
existing vessels was promoted 2- to 3-fold by either T(4) or T(3) at
10(-8)-10(-7) M total hormone concentrations. In the present studies, omolar
concentrations of 3,5-diiodothyropropionic acid (DITPA), a thyroid hormone
analog with inotropic but not chronotropic properties, exhibited potent
proangiogenic activity that was comparable to that obtained with T(3) and T(4)
in both the CAM model and in an in vitro three-dimensional human microvascular
endothelial sprouting assay. The proangiogenesis effect of DITPA was inhibited
by tetraiodothyroacetic acid, a thyroid hormone analog that competes with T(4)
and T(3) for a novel cell surface hormone receptor site on integrin alphavbeta3.
The thyroid hormone analogs DITPA, T(4), and T(4)-agarose, as well as basic
fibroblast growth factor (b-FGF) and vascular endothelial cell growth factor,
demonstrated comparable proangiogenic effects in the CAM model and in the
three-dimensional human microvascular endothelial sprouting model. The
proangiogenesis effect of either DITPA or b-FGF was blocked by PD 98059, an
inhibitor of the ERK1/2 signal transduction cascade. Additionally, a specific
integrin alphavbeta3 small molecule antagonist, XT199, effectively inhibited the
proangiogenesis effect of DITPA and b-FGF. Thus, the proangiogenesis actions of
thyroid hormone and its analog DITPA are initiated at the plasma membrane,
apparently at integrin alphavbeta3, and are MAPK dependent. Previous studies from our laboratory have demonstrated that thyroid hormones
play a key role in cancer progression. In addition, a deaminated form,
tetraiodothyroacetic acid (tetrac), that antagonizes the proliferative action of
these hormones was found to possess anti-cancer functions through its ability to
inhibit cellular proliferation and angiogenesis. The present study was
undertaken to investigate whether tetrac could also suppress the development of
drug resistance, known as a causative factor of disease relapse. Tetrac was
shown to enhance cellular response in vitro to doxorubicin, etoposide,
cisplatin, and trichostatin A in resistant tumor cell lines derived from
neuroblastoma, osteosarcoma, and breast cancer. The mechanism of action of
tetrac did not involve expression of classical drug resistance genes. However,
radiolabeled doxorubicin uptake in cells was enhanced by tetrac, suggesting that
one or more export mechanisms for chemotherapeutic agents are inhibited. Tetrac
was also found to enhance cellular susceptibility to senescence and apoptosis,
suggesting that the agent may target multiple drug resistance mechanisms. Tetrac
has previously been shown to inhibit tumor cell proliferation in vitro. In vivo
studies reported here revealed that tetrac in a pulsed-dose regimen was
effective in suppressing the growth of a doxorubicin-resistant human breast
tumor in the nude mouse. In this paradigm, doxorubicin-sensitivity was not
restored, indicating that (1) the in vitro restoration of drug sensitivity by
tetrac may not correlate with in vivo resistance phenomena and (2) tetrac is an
effective chemotherapeutic agent in doxorubicin-resistant cells. Thyroid hormones [predomitly 3, 5, 3 -I- iodothyronine (T3)] regulate
cholesterol and lipoprotein metabolism but cardiac effects restrict their use as
hypolipidemic drugs. New molecules have been developped which target
specifically the thyroid hormone receptor ss, predomit isoform in liver. The
first thyroid hormone agonist, called GC1, has selective actions compared to T3.
In animals, GC1 reduced serum cholesterol and serum triglycerides, probably by
stimulation important steps in reverse cholesterol transport. Other selective
thyromimetic, KB- 2115 and KB - 141 have similar effects. Another class of
thyroid hormone analogs, the thyronamines have emerged recently but the basic
biology of this new class of endogenous thyroid hormone remains to better
understood. Therefore, these molecules may be a potentially treatment for
obesity and reduction cholesterol, triglycerides and lipoprotein (a). To date
the studies in human are preliminary. Tolerance and efficacy of these drugs are
still under investigation. BACKGROUND: Tetraiodothyroacetic acid (tetrac) is a deaminated analogue of
L-thyroxine that blocks the actions of L-thyroxine and
3,5,3'-triiodo-L-thyronine at the cell surface receptor for thyroid hormone on
integrin alpha v beta 3. Tetrac blocks the proliferative effects of thyroid
hormone on tumor cells and the proangiogenesis actions of the hormone. In the
absence of thyroid hormone, tetrac also blocks angiogenesis induced by various
growth factors. Covalently linked to poly(lactide-co-glycolide), tetrac
oparticles (tetrac NP) do not gain access to the cell interior and act
exclusively at the integrin receptor. Here, the activity of tetrac and tetrac NP
against follicular thyroid carcinoma (FTC)-236 cells was studied in two models:
(1) tumor cell implants in the chick chorioallantoic membrane (CAM) system and
(2) xenografts in the nude mouse.
METHODS: FTC-236 cells (10(6)) were implanted in the CAM (n = 8 each for
control, and for tetrac and tetrac NP, both at 1 microg/CAM) and the actions of
tetrac and tetrac NP were determined after 8 days on tumor-related angiogenesis
and tumor growth. Xenografts of 10(7) FTC-236 cells were implanted in nude mice
(n = 8 per group). Tetrac or tetrac NP was administered intraperitoneal (1 mg/kg
and 1 mg tetrac equivalent/kg, respectively) every other day for 32 days
beginning on day 10, when tumor volume was 200-250 mm(3). Animals were monitored
after discontinuation of treatment up to day 40.
RESULTS: In the CAM paradigm, tetrac and tetrac NP arrested tumor-related
angiogenesis and tumor growth. In the xenograft model, tetrac and tetrac NP
promptly and progressively reduced tumor volume (p < 0.01) over 32 days. There
was some regrowth of tumor after interruption of tetrac treatment, but at day
40, tumor volume and tumor weight at sacrifice were 45-55% below those of
controls (p < 0.01). Animal weight gain was comparable in the control and
treatment groups of animals.
CONCLUSIONS: Tetrac and tetrac NP effectively arrest FTC-236 cell tumor growth
in the CAM and xenograft models, suggesting its potential utility against FTC. OBJECTIVE: The monocarboxylate transporter 8 (MCT8; SLC16A2) has a pivotal role
in neuronal triiodothyronine (T(3)) uptake. Mutations of this transporter
determine a distinct X-linked psychomotor retardation syndrome
(Allan-Herndon-Dudley syndrome (AHDS)) that is attributed to disturbed thyroid
hormone levels, especially elevated T(3) levels. We describe the genetic
analysis of the MCT8 gene in a patient suspected for AHDS and the clinical and
endocrine effects of L-thyroxine (LT(4)) or liothyronine (LT(3)) treatment
intending to overcome the T(3) uptake resistance through alternative
transporters.
METHODS: The six exons of the MCT8 gene were amplified individually by PCR. As
multiple exons were missing, the length of the X-chromosomal deletion was
determined by a dense SNP array, followed by PCR-based fine mapping to define
the exact borders of the deleted segment. The clinical and endocrine data of the
patient during 6.5 years of LT(4) treatment and two periods (3 months each) of
low- and high-dose LT(3) were evaluated.
RESULTS: A partial deletion of the MCT8 gene (comprising five of six exons) was
detected, confirming the suspected AHDS. MCT8 dysfunction was associated with
partial resistance to T(3) at the hypothalamus and pituitary level, with normal
responsiveness at the peripheral organs (liver and cardiovascular system).
Thyroid hormone administration had no beneficial effect on the neurological
status of the patient.
CONCLUSION: We identified a 70 kb deletion encompassing exons 2-6 of the MCT8
gene in our AHDS patient. Both LT(4) and LT(3) administration had no therapeutic
effect. Alternatively, treatment of AHDS patients with thyroid hormone analogs
should be considered. BACKGROUND: The effectiveness of short-term 3,5,3'-triiodothyroacetic acid
(TRIAC) therapy for the treatment of hyperthyroidism caused by thyroid hormone
resistance (RTH) has been documented. Here, we report a 3-year course of TRIAC
therapy in an RTH boy, with a quantitative evaluation of the therapeutic effects
and pharmacological study of TRIAC.
PATIENT FINDINGS: The gene encoding the thyroid hormone receptor beta (THRB) of
the patient carries a P453T mutation. During treatment with up to 3.0 mg TRIAC
per day, reduction in the thyroid volume, resolution of supraventricular
arrhythmia, and decrease in thyroid-stimulating hormone (TSH) and free-thyroxine
(FT4) levels were achieved. In addition, attention-deficit hyperactivity
disorder (ADHD) symptoms improved, with a concomitant decline in the ADHD Rating
Scale score.
SUMMARY: A TRIAC pharmacokinetic study, conducted using triiodothyronine level
as a surrogate for TRIAC level, demonstrated that TRIAC disappears from the
circulation rapidly and has a shorter duration of TSH secretion inhibitory
effect in the RTH patient compared to that in the control subject. Studies of
TSH and FT4 levels over a period of 3 years indicated that the TRIAC effect is
dose dependent.
CONCLUSIONS: TRIAC was effective and safe in ameliorating the effects of
hyperthyroidism and ADHD symptoms in a child with known genetic RTH. Further, it
was demonstrated that TRIAC has a short half-life and functions dose
dependently. |
Are patients with marfan syndrome at increased risk of arrhythmias? | Patients with marfan syndrome carry increased risk for arrhythmias | OBJECTIVES: We sought to assess outcomes in a series of young patients with
Marfan syndrome and to define the prevalence of ventricular arrhythmias in this
patient population.
BACKGROUND: While sudden death is a well-recognized outcome in Marfan syndrome,
ventricular arrhythmias are not well described.
METHODS: Patients were followed with echocardiography, electrocardiography, and
ambulatory electrocardiography. The prevalence and associated factors for
ventricular dysrhythmias were defined.
RESULTS: Seventy patients with Marfan syndrome diagnosed at birth to 52 years
were followed for a period of up to 24 years. All patients had cardiovascular
involvement and were started on medical therapy. No patient died from aortic
dissection, while 4% died from arrhythmias. Ventricular arrhythmias were present
in 21% and were associated with increased left ventricular size, mitral valve
prolapse, and abnormalities of repolarization.
CONCLUSIONS: Cardiac complications are rare in young patients with Marfan
syndrome receiving medical therapy and close clinical follow-up. Sudden death
still occurs, and appears more common in patients with a dilated left ventricle.
Left ventricular dilation may predispose to alterations of repolarization and
fatal ventricular arrhythmias. Marfan's syndrome is a common connective tissue disease with different
musculoskeletal, ophthalmic and cardiac manifestations. Marfan's patients carry
increased risk for cardiac arrhythmias. Only three cases of atrial flutter in
Marfan's patients are described in the literature. We report a fourth case of a
young Marfan's patient who presents with typical atrial flutter after motor
vehicle accident. After electrical cardioversion, sinus rhythm was restored but
he had recurrent atrial flutter on follow up. The patient then underwent
electrophysiological study and successful radiofrequency catheter ablation of
the flutter circuit. Since discharge, the patient has had no documented
arrhythmias on follow up. BACKGROUND: Marfan syndrome (MFS) is a variable, autosomal-domit disorder of
the connective tissue. In MFS serious ventricular arrhythmias and sudden cardiac
death (SCD) can occur. The aim of this prospective study was to reveal
underlying risk factors and to prospectively investigate the association between
MFS and SCD in a long-term follow-up.
METHODS: 77 patients with MFS were included. At baseline serum N-terminal
pro-brain natriuretic peptide (NT-proBNP), transthoracic echocardiogram, 12-lead
resting ECG, signal-averaged ECG (SAECG) and a 24-h Holter ECG with time- and
frequency domain analyses were performed. The primary composite endpoint was
defined as SCD, ventricular tachycardia (VT), ventricular fibrillation (VF) or
arrhythmogenic syncope.
RESULTS: The median follow-up (FU) time was 868 days. Among all risk
stratification parameters, NT-proBNP remained the exclusive predictor (hazard
ratio [HR]: 2.34, 95% confidence interval [CI]: 1.1 to 4.62, p=0.01) for the
composite endpoint. With an optimal cut-off point at 214.3 pg/ml NT-proBNP
predicted the composite primary endpoint accurately (AUC 0.936, p=0.00046,
sensitivity 100%, specificity 79.0%). During FU, seven patients of Group 2
(NT-proBNP ≥ 214.3 pg/ml) reached the composite endpoint and 2 of these patients
died due to SCD. In five patients, sustained VT was documented. All patients
with a NT-proBNP<214.3 pg/ml (Group 1) experienced no events. Group 2 patients
had a significantly higher risk of experiencing the composite endpoint
(logrank-test, p<0.001).
CONCLUSIONS: In contrast to non-invasive electrocardiographic parameter,
NT-proBNP independently predicts adverse arrhythmogenic events in patients with
MFS. |
What are the treatments of choice for GIST (gastrointestinal stromal tumor)? | The surgical resection is a treatment of choice for gastrointestinal stromal tumors. It has been shown that adequate surgical resection correlates with high 5-years survival rates for patients with gastric GIST. When they are localized, the treatment of choice is surgical excision, but advanced tumors have a limited response to chemo or radiotherapy. Imatinib (STI571 or Glivec) is a selective inhibitor or tyrosine kinase proteins that has been used successfully in the treatment of advanced GIST. | Hepatic and peritoneal metastases are the most frequent metastatic lesions in
patients with gastrointestinal stromal tumors (GIST), and may result in intra-
or extrahepatic cholestasis and altered drug metabolism. While the tyrosine
kinase inhibitor imatinib, which has been recently shown to represent the
treatment of choice for GIST, is primarily metabolized by the liver, data on the
pharmacokinetics and the tolerability of imatinib in patients with increased
cholestasis parameters are not yet available. We here report on two patients who
received imatinib in the presence of increased bilirubin and/or cholestasis
parameters. With a follow-up duration of 3-4 months, we observed no toxicities
outside of well-known side effects including some degree of myelosuppression and
fluid retention. This report may aid in the decision of imatinib being given
under close surveillance to this kind of patients. BACKGROUND: Tyrosine kinase inhibitor (Gleevec or STI-571) must be considered
the treatment of choice for metastatic gastrointestinal stromal tumors (GISTs).
The purpose of this article is to address and illustrate a long-term follow-up
of computed tomographic (CT) radiologic findings in patients with metastases
from GIST after Gleevec treatment.
METHODS: We performed a retrospective review of seven patients (four male, three
female) with unresectable metastases from GIST who were treated with STI-571 in
a 1-year period. Patients were followed every 2-4 months by contrast-enhanced CT
for up to 12 months. The size and attenuation of hepatic and peritoneal
metastases on CT were measured and correlated.
RESULTS: Hepatic metastases from GISTs showed significant decreased attenuation
from a mean of 60 HU to a mean of 32 HU (p < 0.01) in the first 2 months and
continued decreasing attenuation to 23 HU at the 12-month follow-up. These
metastases superficially resembled simple cysts. Most metastases became smaller,
with more defined borders, after treatment. Histologic examination in a resected
specimen revealed hepatic cyst with no residual tumor cells, regression of
omental lesions, and extensive necrosis.
CONCLUSIONS: CT findings of unresectable hepatic and peritoneal metastases from
GIST displayed decreasing, near cystic attenuation and size as an effective
regression in response to STI-571 treatment. BACKGROUND: With the introduction of molecularly targeted therapy for
gastrointestinal stromal tumors (GISTs), it became important to distinguish
GISTs from leiomyosarcomas (LMSs). The authors sought to characterize the
clinicopathologic features of these tumors in pediatric patients.
METHODS: The authors reviewed the medical records of 11 patients for whom GIST
or LMS was diagnosed between March 1962 and July 2002 at St. Jude Children's
Research Hospital and reclassified the tumors according to current histologic
and immunophenotypic criteria. The authors also reviewed the literature
pertaining to pediatric GISTs and LMSs.
RESULTS: Seven patients had GISTs, and four had LMS. The median age of the
patients at diagnosis was 11.5 years. At diagnosis, metastases were present in
one patient with GISTs and in another with LMS. Unlike the focal distribution of
CD117 (KIT) in LMS, diffuse and strong immunostaining was observed in GISTs.
Only GISTs expressed CD34. Six patients underwent complete resection (four with
GISTs and two with LMS), four patients underwent incomplete resection (three
with GISTs and one with LMS), and one patient (with LMS) underwent a biopsy
only. Radiotherapy or chemotherapy was used to treat one patient with GISTs and
three patients with LMS. One patient with a high-risk GIST (largest dimension of
32 cm and high mitotic count) was treated with adjuvant imatinib mesylate
outside the preferred setting of a clinical trial, due to concerns regarding the
high risk of tumor recurrence. Four patients with GISTs and two with LMS
survived median disease-free a median of 10.4 years and 4.3 years after
diagnosis, respectively. Tumors in all but one survivor were completely
resected.
CONCLUSIONS: KIT staining helped to distinguish GISTs from LMSs. Surgery was the
treatment of choice for both entities, and tumor resectability was a key
prognostic factor. In the past, numerous reports have advocated primary surgical resection as a
treatment of choice for bleeding gastric stromal tumors (GIST). There were
scarce reports on primary hemostasis with endoscopic therapy. We encountered two
patients who presented to our unit with acute bleeding from gastric fundal
stromal tumor, in whom we were able to achieved successful endoscopic hemostasis
with hemoclip therapy. We describe the management of these cases and reviewed
the current management strategies in bleeding gastric stromal tumor. We report two cases of postoperative recurrence of gastrointestinal stromal
tumor (GIST) treated by the tyrosine kinase inhibitor imatinib mesylate (IM),
and discuss some important items. Case 1: This 63-year-old Japanese man received
a partial gastrectomy for leiomyosarcoma in 1993. Partial hepatectomy and
proximal gastrectomy were performed for liver metastasis and local recurrence in
2001. However, 5 months after surgery, a CT scan showed multiple tumors in the
liver, lung and thyroid. The patient was treated with 300 mg of IM once daily
with transient grade 2 neutropenia and intestinal bleeding. Though the response
to treatment was SD-PR initially, a CT scan 15 months after initial treatment
demonstrated the regrowth of the tumor in his liver. Case 2: A 63-year-old
Japanese woman was treated with 200 mg of IM once daily for multiple liver
metastases after gastrectomy for GIST with grade 3 neutropenia and edema of
legs. The response to treatment was SD, and continued for 12 months. IM is the
treatment of choice for unresectable recurrence of GIST. However, some problems
remained. Both basic and clinical research is necessary to increase the
therapeutic efficacy of IM. BACKGROUND: Gastrointestinal stromal tumours (GISTs) are the most common
mesenchymal tumours of the gastrointestinal tract. They are positive to C-kit
(CD 117), more common in the older males, and mostly in the stomach, less in the
colon and rectum and oesophagus. Benign tumours are more common than the
maligt ones. Classification of GISTs is based on morphology and
immunochemistry.
METHODS: We report two cases of colorectal gastrointestinal mesenchymal tumours,
one on the transverse colon which was found to be immunohistochemically
leiomyosarcoma and the other on the rectum which met the GIST criteria. The
patients underwent transversectomy and abdominal perineal resection,
respectively.
CONCLUSIONS: They did not receive Imanitib postoperatively and two years after
there is no evidence of recurrence. Surgery is the treatment of choice for
resectable GISTs, and other mesenchymal tumours benign or maligt. Gastric lymphoma and gastrointestinal stromal tumours (GISTs) are rare
maligcies of the upper gastrointestinal tract. The most common gastric
lymphoma are low-grade marginal zone B-cell lymphoma (MZBCL) of MALT type. They
develop as a consequence of chronic Helicobacter pylori infection, the
histological hallmark are lymphoepithelial lesions. In early stages of disease,
H. pylori eradication alone may lead to complete lymphoma remission in up to 75%
of cases. Nonresponder or locally advanced lymphoma should be treated with
radiation therapy. Advanced lymphoma may be treated with the nucleoside analogon
cladribine within clinical trials. Based on clinical and novel molecular markers
a risk stratification and a prediction of response to therapy might be possible
in the future. GISTs are mesenchymal tumours that characteristically express
CD-117 (c-kit). They are mostly localized in the upper gastrointestinal tract
and are frequently diagnosed in an advanced stage. Conventional chemotherapy is
ineffective. For resectable non-metastasized tumours surgical therapy is the
treatment of choice. Imatinib is the first and so far only effective systemic
therapy which is presently indicated in irresectable or metastasized GISTs. More
than 80% of patients respond to imatinib therapy either with partial remission
or stable disease. FDG-PET plays an important role in the early prediction of
response to imatinib therapy. The optimal dosage and duration of treatment and
the role of imatinib as adjuvant or neo-adjuvant therapy for GISTs remains to be
defined. Surgical resection is the treatment of choice for the gastrointestinal stromal
tumors (GISTs). In the literature, the 5-year patient survival after surgical
resection, ranged from 48 to 80%, before the era of imatinib mesylate and the
exploration of the prognostication criteria. Imatinib mesylate targets an
intracellular signaling molecule of the natural history and maligt
development of GISTs, and increased the 5-year survival rate, after the
resection of primary low-risk GISTs, to similar values to the normal population.
For high-risk GISTs, current knowledge which is still under expansion, show
major improvement at the 1-year survival rate of more than 90% versus less than
50% before imatinib era. After surgical resection, for both low and high
maligt potential GISTs, a closed control directed to the early identification
of confined resectable recurrences, is required. This paper assesses the current
knowledge of GIST management, motivated by a case of patient with intermediate
risk GIST. There is now considerable interest in gastrointestinal stromal tumor (GIST)
because it can be treated effectively with a targeted molecular agent. The
majority of GISTs contain an activating mutation in the KIT protooncogene or,
occasionally, in the platelet-derived growth factor-alpha (PDGFRA) gene. Five
years ago, imatinib mesylate, a specific molecular inhibitor of the protein
products of these 2 genes, was applied to metastatic GIST. Approximately 80% of
patients with metastatic GIST benefit from imatinib, although acquired
resistance to the agent may develop. For patients with primary GIST, surgery
remains the treatment of choice, and whether outcome is improved by adjuvant
imatinib is currently under broad investigation. A combination of imatinib
therapy and surgery also may be effective in a subset of patients with
metastatic or unresectable primary GIST. In this review, the authors summarize
the new multimodality approach to GIST. The integration of surgery and molecular
therapy in GIST will serve as a prototype for the management of other solid
tumors for which targeted agents become available. Gastrointestinal stromal tumors (GIST) comprise a rare group of neoplasms of
unpredictable maligt. The stomach is the most common site of occurrence in
the gastrointestinal tract. A combination of prognostic factors (patient age,
histologic grade, mitotic rate, tumor size, and DNA analysis) have been utilized
to predict their biologic behavior. Lymphatic spread of gastrointestinal stromal
tumors is uncommon therefore a formal lymph node dissection is not standard
surgical management. Consequently, complete surgical resection of the primary
tumor is the most definitive treatment. Although numerous operative approaches
have been utilized for gastric submucosal tumors, laparoscopic wedge resection
has been regarded as the treatment of choice in recent years. As widespread use
of diagnostic endoscopy has exposed a number of gastric submucosal tumors, the
laparoscopic wedge resections are being performed with increased frequency. In
this article, we describe a successfully performed laparoscopic sleeve
gastrectomy for a bleeding GIST. OBJECTIVES: The aim of the study was the analysis of women with gastrointestinal
stromal tumors (GIST) of small intestine treated and followed-up in Cancer
Center-Institute in Warsaw, who were primary operated in gynecological
departments due to suspicion of gynecological neoplasm.
MATERIALS AND METHODS: In the database of Clinical GIST Registry from 2001 to
2004 we identified 44 women with the diagnosis of CD117(+) GIST of small
intestine, what corresponds to 34% (44/130) all female GIST patients. Sixteen of
them (36.4%, 16/44) were primary operated on in gynecological departments due to
the tentative diagnosis of gynecological neoplasm.
RESULTS: The only indication for operation in 29 women was undiagnosed
microscopically tumor of the pelvis. Sixteen of them (55.2%, 16/29) were
operated on schedule in gynecological departments. The others 15 patients were
operated due to: ileus and perforation of digestive tract (8), gastrointestinal
bleeding (3), abdominal pain (2) and others (2). In analyzed group of patients
20 women (45.5%) after GIST excision remain without evidence of disease with
median follow-up time of 9 months, and in 24 patients (54.5%) GIST recurred in
median time of 18.5 months. In this latter group 23 patients were treated with
imatinib due to inoperable/metastatic lesions. Estimated 2-year overall survival
(calculated form the date of imatinib introduction) was 75%.
CONCLUSIONS: GISTs, especially of small intestine, may simulate in women
gynecological tumor, particularly of the ovary. Radical surgery remains the most
effective method of GIST treatment. In inoperable/metastatic lesion the
treatment of choice is tyrosinase kinase inhibitor--imatinib. Maligt gastointestinal stromal tumors (M-GIST) are rare mesenchymal tumors
that arise in the wall of the gastrointestinal (GI) tract. Small intestinal GIST
account for approximately 35% of all GIST the diagnosis of these tumors is
difficult to establish, because the symptoms are vague and non-specific and
traditional endoscopy is commonly unsatisfactory. Because of the infrequent
nature of maligt small bowel tumors, the natural history and factors
affecting outcome remain poorly defined; stage at presentation and complete
surgical resection seem to be the main prognostic factors. For these rare
tumors, surgery remains the treatment of choice, with little efficacy reported
for irradiation, chemiotherapy, or both. Two cases of GIST of the jejunum,
complicated by intestinal bleeding and intestinal obstruction respectively, are
presented and a review of the literature is made. Gastrointestinal stromal tumours are the most common mesenchimal tumours of the
gastrointestinal tract. Diagnosis of these tumours is difficult to establish,
because symptoms are vague and traditional diagnostic tests are not specific.
Natural history remains poorly defined and many criteria have been correlated
with prognosis; for instance, some authors have found that GIST localization
influences clinical behavior, that has not been confirmed by other authors;
actually, tumour size, mitotic rate and complete resection seem to be the main
prognostic factors. Surgical resection is the treatment of choice, with little
efficacy reported for irradiation, conventional cytotoxic agents or both.
Nevertheless, imatinib mesylate has recently demonstrated significant activity
and tolerability in the treatment of maligt unresectable or metastatic GIST.
Three cases of GIST, complicated by intestinal bleeding and intestinal
obstruction respectively, are presented and a review of the literature is made. BACKGROUND: Non-islet cell tumor induced hypoglycemia (NICTH) is a very rare
phenomenon, but even more so in gastrointestinal stromal tumors. It tends to
present in large or metastatic tumors, and can appear at any time in the
progression of the disease. We present herein a case of NICTH in a GIST tumor
and report an exon 9 mutation associated to it.
CASE PRESENTATION: A thirty nine year-old man with a recurrent, metastatic
gastrointestinal stromal tumor presented to the hospital with nausea, dizziness,
loss of consciousness, and profound hypoglycemia (20 mg/dL). There was no
evidence of factitious hypoglycemia. He was stabilized with a continuous glucose
infusion and following selective vascular embolization, the patient underwent
debulking of a multicentric 40 cm x 25 cm x 10 cm gastrointestinal stromal
tumor. After resection, the patient became euglycemic and returned to his normal
activities. Tumor analysis confirmed excessive production of insulin-like growth
factor II m-RNA and the precursor protein, "big" insulin-like growth factor II.
Mutational analysis also identified a rare, 6 bp tandem repeat insert (gcctat)
at position 1530 in exon 9 of KIT.
CONCLUSION: Optimal management of gastrointestinal stromal tumor-induced
hypoglycemia requires a multidisciplinary approach, and surgical debulking is
the treatment of choice to obtain immediate symptom relief. Imatinib or
combinations of glucocorticoids and growth hormone are alternative palliative
strategies for symptomatic hypoglycemia. In addition, mutations in exon 9 of the
tyrosine kinase receptor KIT occur in 11-20% of GIST and are often associated
with poor patient outcomes. The association of this KIT mutation with non-islet
cell tumor induced hypoglycemia has yet to be established. Gastrointestinal stromal tumors (GISTs) represent the most common mesenchymal
neoplasms of the GI tract. The optimal management of GISTs has been evolving
rapidly over the past 5 years and depends on proper histopathologic and
radiologic diagnosis as well as appropriate multidisciplinary medical and
surgical treatments. Complete surgical resection of primary localized GIST with
negative margins remains the best therapeutic option today. In the setting of
locally advanced or metastatic disease, imatinib mesylate has emerged as the
initial treatment of choice, administered either as cytoreductive or as
definitive treatment. Surgery or ablative modalities in this setting are
becoming increasingly employed, particularly when all disease becomes amenable
to gross resection or destruction, or to manage complications arising from the
disease following imatinib failure. We report on the surgical management of an
unusual and clinically significant complication following progression of disease
secondary to imatinib resistance. The role of surgical therapy in the management
of GIST complications following resistance to imatinib and the integration of
surgical and molecular therapy of locally advanced or metastatic GISTs are
discussed. Gastrointestinal stromal tumors (GISTs) rarely arise in the rectum. Whereas a
local resection with negative margins is generally considered adequate for
resectable GISTs, a wide resection is usually indicated for rectal lesions
because of the technical impossibility of local resection. We report the cases
of two patients who underwent resection of a rectal GIST using a transsacral
approach. Both patients had an uneventful postoperative course, and no evidence
of recurrence has been identified. The transsacral approach appears to be less
invasive and should be considered as the treatment of choice for a rectal GIST. Gastrointestinal stromal tumours are rare neoplasms originating from the
connective tissue of the digestive tract and constitute most of the
non-epithelial primitive digestive tumours. The origin from the interstitial
cell of Cajal is appreciated because of this tumours constantly present the
expression of the surface antigens CD34 and CD 117 which can be determined
immunohistochemistry. In the majority of cases, GISTs are symptomatic and
symptoms are most commonly related to mass effect or bleeding. Asymptomatic
GISTs are often found incidentally on physical examination, radiologic imaging,
endoscopy, laparotomy or laparoscopy. US endoscopy and fine needle aspiration
with subsequent immunohistochemistry analysis afford the best diagnostic
accuracy. In primary and localized GISTs surgery is always indicated and
laparoscopic technique is feasible and is recommended as the treatment of choice
for all the patients. Imatinib should be started in metastatic or recurrent
disease and neoadjuvant imatinib is also experimental, although its use may be
justified in unresectable or marginally resectable GIST. Sunitinib has recently
been approved for patients with GIST principally those who fail imatinib
therapy. Our experience is based on the study of 7 GISTs: only in 2 cases the
neoplasm was found occasionally; in the other, symptoms were related to mass
effect or bleeding. Laparoscopic tumour resection was then performed in all the
patients. The definitive diagnosis of gastrointestinal stromal tumour, was made
postoperatively by analysis of the histopathological and immunohistochemical
findings. We confirmed constant high positivity for CD34 and for CD117. Even in
the absence of unfavourable prognostic indicators, all patients are regularly
followed-up. Background. Surgery remains the only curative treatment for gastrointestinal
stromal tumour (GIST). Resection needs to ensure tumour-free margins while
lymphadenectomy is not required. Thus, partial gastric resection is the
treatment of choice for small gastric GISTs. Evidence on whether performing
resection laparoscopically compromises outcome is limited. Methods. We compiled
patients undergoing laparoscopic resection of suspected gastric GIST between
2003 and 2007. Follow-up was performed to obtain information on tumour
recurrence. Results. Laparoscopic resection with free margins was performed in
21/22 patients. Histology confirmed GIST in 17 cases, 4 tumours were benign
neoplasms. Median operation time and postoperative stay for GIST patients were
130 (range 80-201) mins and 7 (range 5-95) days. Two patients experienced
stapler line leakage necessitating surgical revision. After median follow-up of
18 (range 1-53) months, no recurrence occurred. Conclusions. Laparoscopic
resection of gastric GISTs yields good perioperative outcomes. Oncologic outcome
needs to be assessed with longer follow-up. For posterior lesions, special
precaution is needed. Laparoscopic resection could become standard for
circumscribed gastric GISTs if necessary precautions for oncological procedures
are observed. INTRODUCTION: Gastrointestinal stromal tumors are the commonest mesenchymal
tumors of the gastrointestinal tract, the stomach and small intestine are the
favored sites of occurrence. They rarely occur in the colon, rectum and
esophagus. GIST is neoplasm of mesenchymal origin originating from precursors of
the interstitial cells of cajal. The symptoms of gastrointestinal stromal tumor
depend on the site and size of the tumor, and may include abdominal pain,
gastrointestinal bleeding or signs of obstruction; small tumors may, however, be
asymptomatic. Majority of the patients with gastrointestinal stromal tumor have
bloody stools and abdominal pain as the commonest manifestation. We describe a
young female with extra luminal colonic gastrointestinal stromal tumor
presenting as mass abdomen.
CASE PRESENTATION: We describe 34-year-old female from north Indian state of
Jammu and Kashmir who had presented with history of slowly increasing epigastric
lump associated with abdominal discomfort of 4 months duration. She had no
features of luminal obstruction. Her contrast enhanced computed tomography
abdomen revealed a large extra-colonic mass in relation to transverse colon. The
tumor was resected and histology was suggestive of gastrointestinal stromal
tumor.
CONCLUSION: Extra luminal colonic gastrointestinal stromal tumors are very rare
and can present as mass abdomen. Resection is the treatment of choice. Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the alimentary
tract rarely observed in children. The treatment of choice for GIST is surgical
resection. Although the prognosis of GISTs with low maligt potential is
excellent, high-maligt-potential GISTs have a high rate of recurrence.
Prognostic factors such as tumor size, mitotic rate and presence of metastases
may provide an indication for adjuvant imatinib mesylate (IM) treatment. Here we
present a young patient with a large GIST with high-risk features who is in
complete remission after surgical excision and adjuvant IM treatment. This
patient is the only colon-located CD117-positive case where IM was administered.
The exact indications as well as the optimal dose and duration of IM need to be
clarified with the contribution of new cases and the growing experience of this
rare disease. BACKGROUND: Gastrointestinal stromal tumours (GISTs) are the most common
mesenchymal tumours of the gastrointestinal tract and make up 1-2% of all
gastrointestinal maligcies. Traditionally, the treatment of choice for
primary disease is surgical resection; however, no single surgeon or institution
gets extensive exposure to these patients so appropriate decision-making is
difficult, particularly since the introduction of the tyrosine kinase inhibitor
imatinib, which has become an important additional management tool.
METHOD: All patients were diagnosed and treated for GISTs in Christchurch
Hospital (Christchurch, New Zealand) between 1 January 2000 and 31 December
2010. We maintain a prospective database of all patients with GISTs. Data on
clinical and histopathological variables, management and survival outcomes were
recorded. These were then reviewed.
RESULTS: There were 93 patients in this study. Fifty were women. Median age was
69 (interquartile range (IQR) 59-76) years. Fifty-one tumours were located in
the stomach, 27 in the small bowel, six in the colon, three in the oesophagus,
one in the rectum and five were extra-gastrointestinal. In total, 22 patients
received imatinib therapy; four patients with metastatic disease had imatinib as
sole therapy. The median follow-up was 58 (IQR 30-90) months. The 5-year overall
survival and disease-free survival (DFS) for the entire study population was 69%
and 64%, respectively. The 5-year DFS was higher for all patients who have
localized disease when compared with those who have metastatic disease (76%
versus 28%, P-value 0.001).
CONCLUSION: Surgery aiming at an R0 resection remains the mainstay of treatment.
We propose the most effective way to grow the knowledge base in New Zealand is
the establishment of a national register, thereby allowing better clinical
decision-making by interpretation of a larger data set. |
List Kartagener Syndrome Triad. | The triad of situs inversus, bronchiectasis and sinusitis is known as Kartagener syndrome. | A case of a nine year and eight months old child with Kartagener's syndrome
(triad) is described: chronic maxillary sinusitis, bronchiectasis and "situs
inversus totalis". Literature is reviewed. It is important to understand
genetical and etiology aspects of bronchiectasis. Diagnosis was considered on
the basis of physical and radiographic examination. Ear, nose and throat symptoms and signs were studied in 15 patients with
Kartagener's syndrome: a triad consisting of chronic rhinosinusitis, chronic
bronchitis with bronchiectasis, and situs inversus. The triad is caused by
primary ciliary dyskinesia and characterized by absent or considerably reduced
mucociliary transport. Daily accumulation of secretions in the nose, chronic
recurrent secretory otitis media and sinusitis occurred in all subjects.
Characteristically, nasal discharge started at birth or was first detected in
early childhood. Apparently, the frequency of common colds and of acute purulent
otitis media was not increased. As primary ciliary dyskinesia can occur without
situs inversus, knowledge of the typical ENT symptoms is essential for making an
early diagnosis, which is important for the correct management of the disease.
While a conservative surgical approach to treatment of the ENT symptoms is
recommended, early and active treatment of the bronchial symptoms is probably
important for prevention of further lung damage and development of
bronchiectasis. Kartagener's syndrome is a well known classical triad of presentations
consisting of bronchiectasis, sinusitis and situs inversus. It is now recognized
that the syndrome is an extreme presentation of primary ciliary dyskinesia, a
large group of conditions with ultrastructural ciliary defects, leading to poor
ciliary motility in various organ systems. A case of Kartagener's syndrome is
presented in an eight year old Thai boy in whom the ultrastructural ciliary
defects have been examined and described in detail for the first time in
Thailand. Incomplete lack of dynein arms was recognized. In addition,
disorientation of ciliary axis was noticed. Due to severe bronchiectatic changes
of the right lower lobe and right lingular lobe which did not improve despite
adequate antibiotics, these lobes were surgically removed. The child has done
well since, but still suffers occasional and recurrent bouts of sinusitis. A comprehensive clinicomorphological examination of 24 children with
Zivert-Kartagener syndrome ascertained the complete triad (bronchiectasis,
maldevelopment of the sinuses and transposition of the viscera) in all of them.
Initial signs of the respiratory affection are shown to arise early. The disease
progression depended primarily on the scope of pulmonary damage.
Electron-microscopic findings at examination of the patients' tracheal mucosa
obtained at bronchoscopy revealed reduced number of ciliary cells and abnormal
inner structure of the cilia. The impairment in some patients was focal. The
attention is drawn to the benefit of early diagnosis and follow-up for such
patients who should be treated surgically after careful consideration of
potential risks. The relationship of Kartagener's syndrome to immobile cilia syndrome is a
fascinating merging of clinical observations and basic science in Zurich,
Stockholm, and Toronto. In 1933, Manes Kartagener, a Zurich pulmonary physician,
reported four patients with the triad of sinusitis, bronchiectasis, and situs
inversus. In the following decades, he reviewed reports of hundreds of cases,
but the fact that the male patients with the condition never had offspring
eluded his notice. In the 1970s, Bjorn Afzelius, a Ph.D. ultrastructuralist from
Stockholm, reported cilia immotility in infertile males, some of the cases
occurring in families. Half of the cases had Kartagener's triad. The observation
of Afzelius was soon applied to children by Jennifer Sturgess, a Ph.D.
ultrastructuralist, and her medical colleagues in Toronto. With over 500 MEDLINE
references since 1966 on Kartagener's and over 1,000 references on immotile
cilia, the causes of the pulmonary infections have become clearer as the
patients demonstrate impaired clearance of mucus with resultant sinus and
bronchial disease. The cause of the situs inversus remains elusive to this day.
It is appropriate to call the condition Kartagener-Afzelius syndrome. Kartagener's syndrome is a rare disorder characterized by the triad of situs
inversus, including dextrocardia, bronchiectasis and paranasal sinusitis. We
report the anaesthetic management of a patient with Kartagener's syndrome and
postrenal transplant immunosuppression, presenting for repair of uterovaginal
prolapse. Combined spinal epidural anaesthesia was administered to this patient.
The anaesthetic considerations of this rare disorder and the relative advantages
of the regional technique over general anaesthesia in this situation are
discussed. Kartagener syndrome (KS), an autosomal recessively inherited disease, is
characterized by the triad of situs inversus, bronchiectasis and sinusitis. This
disorder affects the activity of proteins important to the movement of cilia,
especially in the respiratory tract and the spermatozoa, developing a series of
systemic alterations, which can be diagnosed through radiographic examination.
The aim of this paper is to describe a clinical case of this unusual pathology,
including a brief literature review, emphasising the radiographic aspects of
this pathology and stressing the importance of early diagnosis, which could be
determined by an oral radiologist. Kartagener syndrome (a clinical variant of primary ciliary dyskinesia) is a
recessive autossomical disease characterized by the triad of chronic sinusitis,
bronchiectasis and situs inversus with dextrocardia. We report one case
described in a 8 years old boy who besides presented a seromucous otitis and
bronchitis of repetition. Finally we performed a short bibliographic review at
respect of this uncommon pathology. Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disease,
characterized by ciliary disfunction and impaired mucociliary clearance,
resulting in a range of clinical manifestations such as chronic bronchitis,
bronchiectasis, chronic rhino-sinusitis, chronic otitis media, situs viscerum
inversus in almost 40-50% of cases and male infertility. The triad situs
viscerum inversus, bronchiectasis and sinusitis is known as Kartagener syndrome.
Up to now little is known about genetic, diagnostic and therapeutic aspects of
primary motile ciliary diseases in children: for this reason, diagnosis is
generally delayed and almost all treatments for PCD are not based on randomized
studies but extrapolated from cystic fibrosis guidelines. The aim of this review
is to propose to pediatricians a summary of current clinical and diagnostic
evidence to obtain better knoledwge of this condition. The earlier diagnosis and
the right treatment are both crucial to improve the prognosis of PCD. Kartagener's syndrome is a very rare congenital malformation comprising of a
classic triad of sinusitis, situs inversus and bronchiectasis. Primary ciliary
dyskinesia is a genetic disorder with manifestations present from early life and
this distinguishes it from acquired mucociliary disorders. Approximately one
half of patients with primary ciliary dyskinesia have situs inversus and, thus
are having Kartagener syndrome. We present a case of 12 year old boy with
sinusitis, situs inversus and bronchiectasis. The correct diagnosis of this rare
congenital autosomal recessive disorder in early life is important in the
overall prognosis of the syndrome, as many of the complications can be prevented
if timely management is instituted, as was done in this in this case. Primary ciliary dyskinesia (PCD) is a genetic disease that causes abnormalities
in ciliary structure and/or function. Ciliated cells line the upper and lower
respiratory tracts and the Eustachian tube. Impairment of mucus clearance at
these sites leads to sinusitis, repeated pulmonary infections, bronchiectasis,
and chronic otitis media. Situs inversus occurs randomly in approximately 50% of
subjects with PCD. The triad of situs inversus, bronchiectasis and sinusitis is
known as Kartagener syndrome. PCD is usually an autosomal recessive disease, but
occasional instances of X-linked transmission have been reported. Specific
diagnosis requires examination of ciliary function or structure on light and
electron microscopy. Early diagnosis and respiratory management are important in
order to prevent the development of bronchiectasis and deterioration in lung
function. We report early diagnosis of PCD on nasal mucosal biopsy in two
newborns who presented with prolonged respiratory distress and rhinorrhea. BACKGROUND: KARTAGENER SYNDROME (KS) IS A RARE CONGENITAL DISEASE CHARACTERISED
BY A CLINICAL TRIAD OF SYMPTOMS: situs inversus, chronic rhinosinusitis, and
bronchiectasis. Although congenital ciliary defect is recognised as the main
cause of this syndrome, it remains difficult to treat the associated airway
infection.
CASE REPORT: A 17-year-old female patient presented with repeated refractory
airway infection. She also had bronchiectasis and situs inversus. Electron
microscopic evaluation of her nasal mucosa revealed ciliary defect and confirmed
the diagnosis of KS. She underwent functional endoscopic sinus surgery (FESS)
followed by long-term postoperative debridement of the sinonasal cavity. This
treatment reduced chronic rhinosinusitis and protected against subsequent airway
infection in a 7-year follow-up.
CONCLUSION: FESS is effective for relieving both chronic rhinosinusitis and lung
infection of KS in the long term. |
Which disease is associated with the ectopic expression of the protein encoded by the gene DUX4? | Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused by decreased epigenetic repression of the D4Z4 macrosatellite repeats and ectopic expression of DUX4, a retrogene encoding a germline transcription factor encoded in each repeat. | Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal domit disorder
linked to contractions of the D4Z4 repeat array in the subtelomeric region of
chromosome 4q. By comparing genome-wide gene expression data from muscle
biopsies of patients with FSHD to those of 11 other neuromuscular disorders,
paired-like homeodomain transcription factor 1 (PITX1) was found specifically
up-regulated in patients with FSHD. In addition, we showed that the double
homeobox 4 gene (DUX4) that maps within the D4Z4 repeat unit was up-regulated in
patient myoblasts at both mRNA and protein level. We further showed that the
DUX4 protein could activate transient expression of a luciferase reporter gene
fused to the Pitx1 promoter as well as the endogenous Pitx1 gene in transfected
C2C12 cells. In EMSAs, DUX4 specifically interacted with a 30-bp sequence
5'-CGGATGCTGTCTTCTAATTAGTTTGGACCC-3' in the Pitx1 promoter. Mutations of the
TAAT core affected Pitx1-LUC activation in C2C12 cells and DUX4 binding in
vitro. Our results suggest that up-regulation of both DUX4 and PITX1 in FSHD
muscles may play critical roles in the molecular mechanisms of the disease. Facioscapulohumeral muscular dystrophy (FSHD) is a domit disease linked to
contractions of the D4Z4 repeat array in 4q35. We have previously identified a
double homeobox gene (DUX4) within each D4Z4 unit that encodes a transcription
factor expressed in FSHD but not control myoblasts. DUX4 and its target genes
contribute to the global dysregulation of gene expression observed in FSHD. We
have now characterized the homologous DUX4c gene mapped 42 kb centromeric of the
D4Z4 repeat array. It encodes a 47-kDa protein with a double homeodomain
identical to DUX4 but divergent in the carboxyl-terminal region. DUX4c was
detected in primary myoblast extracts by Western blot with a specific antiserum,
and was induced upon differentiation. The protein was increased about 2-fold in
FSHD versus control myotubes but reached 2-10-fold induction in FSHD muscle
biopsies. We have shown by Western blot and by a DNA-binding assay that DUX4c
over-expression induced the MYF5 myogenic regulator and its DNA-binding
activity. DUX4c might stabilize the MYF5 protein as we detected their
interaction by co-immunoprecipitation. In keeping with the known role of Myf5 in
myoblast accumulation during mouse muscle regeneration DUX4c over-expression
activated proliferation of human primary myoblasts and inhibited their
differentiation. Altogether, these results suggested that DUX4c could be
involved in muscle regeneration and that changes in its expression could
contribute to the FSHD pathology. Autosomal domit facioscapulohumeral muscular dystrophy (FSHD) has an unusual
pathogenic mechanism. FSHD is caused by deletion of a subset of D4Z4
macrosatellite repeat units in the subtelomere of chromosome 4q. Recent studies
provide compelling evidence that a retrotransposed gene in the D4Z4 repeat,
DUX4, is expressed in the human germline and then epigenetically silenced in
somatic tissues. In FSHD, the combination of inefficient chromatin silencing of
the D4Z4 repeat and polymorphisms on the FSHD-permissive alleles that stabilize
the DUX4 mRNAs emanating from the repeat result in inappropriate DUX4 protein
expression in muscle cells. FSHD is thereby the first example of a human disease
caused by the inefficient repression of a retrogene in a macrosatellite repeat
array. Double homeobox 4 (DUX4) is a candidate disease gene for facioscapulohumeral
dystrophy (FSHD), one of the most common muscular dystrophies characterized by
progressive skeletal muscle degeneration. Despite great strides in understanding
precise genetics of FSHD, the molecular pathophysiology of the disease remains
unclear. One of the major limitations has been the availability of appropriate
molecular tools to study DUX4 protein. In the present study, we report the
development of five new monoclonal antibodies targeted against the N- and
C-termini of human DUX4, and characterize their reactivity using Western blot
and immunofluorescence staining. Additionally, we show that expression of the
canonical full coding DUX4 induces cell death in human primary muscle cells,
whereas the expression of a shorter splice form of DUX4 results in no such
toxicity. Immunostaining with these new antibodies reveals a differential effect
of two DUX4 isoforms on human muscle cells. These antibodies will provide an
excellent tool for investigating the role of DUX4 in FSHD pathogenesis. BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a domit disease
linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.
Within each repeat unit is a gene, DUX4, that can encode a protein containing
two homeodomains. A DUX4 transcript derived from the last repeat unit in a
contracted array is associated with pathogenesis but it is unclear how.
METHODS: Using exon-based microarrays, the expression profiles of myogenic
precursor cells were determined. Both undifferentiated myoblasts and myoblasts
differentiated to myotubes derived from FSHD patients and controls were studied
after immunocytochemical verification of the quality of the cultures. To further
our understanding of FSHD and normal myogenesis, the expression profiles
obtained were compared to those of 19 non-muscle cell types analyzed by
identical methods.
RESULTS: Many of the ~17,000 examined genes were differentially expressed
(>2-fold, p<0.01) in control myoblasts or myotubes vs. non-muscle cells (2185
and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and
797, respectively). Surprisingly, despite the morphologically normal
differentiation of FSHD myoblasts to myotubes, most of the disease-related
dysregulation was seen as dampening of normal myogenesis-specific expression
changes, including in genes for muscle structure, mitochondrial function, stress
responses, and signal transduction. Other classes of genes, including those
encoding extracellular matrix or pro-inflammatory proteins, were upregulated in
FSHD myogenic cells independent of an inverse myogenesis association.
Importantly, the disease-linked DUX4 RNA isoform was detected by RT-PCR in FSHD
myoblast and myotube preparations only at extremely low levels. Unique insights
into myogenesis-specific gene expression were also obtained. For example, all
four Argonaute genes involved in RNA-silencing were significantly upregulated
during normal (but not FSHD) myogenesis relative to non-muscle cell types.
CONCLUSIONS: DUX4's pathogenic effect in FSHD may occur transiently at or before
the stage of myoblast formation to establish a cascade of gene dysregulation.
This contrasts with the current emphasis on toxic effects of experimentally
upregulated DUX4 expression at the myoblast or myotube stages. Our model could
explain why DUX4's inappropriate expression was barely detectable in myoblasts
and myotubes but nonetheless linked to FSHD. DUX4, a homeobox-containing gene present in a tandem array, is implicated in
facioscapulohumeral muscular dystrophy (FSHD), a domit autosomal disease. New
findings about DUX4 have raised as many fundamental questions about the
molecular pathology of this unique disease as they have answered. This review
discusses recent studies addressing the question of whether there is extensive
FSHD-related transcription dysregulation in adult-derived myoblasts and
myotubes, the precursors for muscle repair. Two models for the role of DUX4 in
FSHD are presented. One involves transient pathogenic expression of DUX4 in many
cells in the muscle lineage before the myoblast stage resulting in a persistent,
disease-related transcription profile ('Majority Rules'), which might be
enhanced by subsequent oscillatory expression of DUX4. The other model
emphasizes the toxic effects of inappropriate expression of DUX4 in only an
extremely small percentage of FSHD myoblasts or myotube nuclei ('Minority
Rules'). The currently favored Minority Rules model is not supported by recent
studies of transcription dysregulation in FSHD myoblasts and myotubes. It also
presents other difficulties, for example, explaining the expression of
full-length DUX4 transcripts in FSHD fibroblasts. The Majority Rules model is
the simpler explanation of findings about FSHD-associated gene expression and
the DUX4-encoded homeodomain-type protein. PURPOSE OF REVIEW: In recent years, we have seen remarkable progress in our
understanding of the disease mechanism underlying facioscapulohumeral muscular
dystrophy (FSHD). The purpose of this review is to provide a comprehensive
overview of our current understanding of the disease mechanism and to discuss
the observations supporting the possibility of a developmental defect in this
disorder.
RECENT FINDINGS: In the majority of cases, FSHD is caused by contraction of the
D4Z4 repeat array (FSHD1). This results in local chromatin relaxation and stable
expression of the DUX4 retrogene in skeletal muscle, but only when a polymorphic
DUX4 polyadenylation signal is present. In some cases (FSHD2), D4Z4 chromatin
relaxation and stable DUX4 expression occur in the absence of D4Z4 array
contraction. DUX4 is a germline transcription factor and its expression in
skeletal muscle leads to activation of early stem cell and germline programs and
transcriptional activation of retroelements.
SUMMARY: Recent studies have provided a plausible disease mechanism for FSHD in
which FSHD results from inappropriate expression of the germline transcription
factor DUX4. The genes regulated by DUX4 suggest several mechanisms of muscle
damage, and provide potential biomarkers and therapeutic targets that should be
investigated in future studies. Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular
dystrophy characterized by an asymmetric progressive weakness and wasting of the
facial, shoulder and upper arm muscles, frequently accompanied by hearing loss
and retinal vasculopathy. FSHD is an autosomal domit disease linked to
chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading
candidate gene as causative of FSHD. However, DUX4 expression is extremely low
in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in
human FSHD. Here, we show that the misexpression of very low levels of human
DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD
patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl)
mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less
pigmentation of the eyes, altered morphology of ears, developmental abnormality
of fin muscle, disorganization of facial musculature and/or degeneration of
trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant
localization of myogenic cells marked with α-actin promoter-driven enhanced
green fluorescent protein outside somite boundary, especially in head region.
These abnormalities were rescued by coinjection of the short form of DUX4
(DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at
extremely low level, can recapitulate the phenotype observed in FSHD patients in
a vertebrate model. These results strongly support the current hypothesis for a
role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during
development is important for the pathogenesis of FSHD. Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of
the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded
DUX4 gene in skeletal muscle. The more common form, autosomal domit FSHD1, is
caused by contraction of the D4Z4 array, whereas the genetic determits and
inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here, we
show that mutations in SMCHD1 (encoding structural maintece of chromosomes
flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein
levels and segregate with genome-wide D4Z4 CpG hypomethylation in human
kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and
a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4
expression. Reducing SMCHD1 levels in skeletal muscle results in D4Z4
contraction-independent DUX4 expression. Our study identifies SMCHD1 as an
epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic
determit of FSHD2 and possibly other human diseases subject to epigenetic
regulation. Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy caused
by decreased epigenetic repression of the D4Z4 macrosatellite repeats and
ectopic expression of DUX4, a retrogene encoding a germline transcription factor
encoded in each repeat. Unaffected individuals generally have more than 10
repeats arrayed in the subtelomeric region of chromosome 4, whereas the most
common form of FSHD (FSHD1) is caused by a contraction of the array to fewer
than 10 repeats, associated with decreased epigenetic repression and variegated
expression of DUX4 in skeletal muscle. We have generated transgenic mice
carrying D4Z4 arrays from an FSHD1 allele and from a control allele. These mice
recapitulate important epigenetic and DUX4 expression attributes seen in
patients and controls, respectively, including high DUX4 expression levels in
the germline, (incomplete) epigenetic repression in somatic tissue, and
FSHD-specific variegated DUX4 expression in sporadic muscle nuclei associated
with D4Z4 chromatin relaxation. In addition we show that DUX4 is able to
activate similar functional gene groups in mouse muscle cells as it does in
human muscle cells. These transgenic mice therefore represent a valuable animal
model for FSHD and will be a useful resource to study the molecular mechanisms
underlying FSHD and to test new therapeutic intervention strategies. Telomeres may regulate human disease by at least two independent mechanisms.
First, replicative senescence occurs once short telomeres generate DNA-damage
signals that produce a barrier to tumor progression. Second, telomere position
effects (TPE) could change gene expression at intermediate telomere lengths in
cultured human cells. Here we report that telomere length may contribute to the
pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a
late-onset disease genetically residing only 25-60 kilobases from the end of
chromosome 4q. We used a floxable telomerase to generate isogenic clones with
different telomere lengths from affected patients and their unaffected siblings.
DUX4, the primary candidate for FSHD pathogenesis, is upregulated over ten-fold
in FSHD myoblasts and myotubes with short telomeres, and its expression is
inversely proportional to telomere length. FSHD may be the first known human
disease in which TPE contributes to age-related phenotype. Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the
subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the
DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD
pathophysiology. However, no one knows what triggers muscle defect and when
alteration arises. To gain further insights into the molecular mechanisms of the
disease, we evaluated at the molecular level, the perturbation linked to the
FSHD genotype with no a priori on disease onset, severity or penetrance and
prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses
carrying a short pathogenic D4Z4 array (n = 6) compared with fetuses with a
non-pathogenic D4Z4 array (n = 21). By measuring expression of several
muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR
and western blotting, we observed a global dysregulation of genes involved in
myogenesis including MYOD1 in samples with <11 D4Z4. The DUX4-fl pathogenic
transcript was detected in FSHD biopsies but also in controls. Importantly, in
FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition,
several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses.
Our study is the first to examine fetuses carrying an FSHD-linked genotype and
reveals an extensive dysregulation of several muscle-specific and 4q35 genes at
early development stage at a distance from any muscle defect. Overall, our work
suggests that even if FSHD is an adult-onset muscular dystrophy, the disease
might also involve early molecular defects arising during myogenesis or early
differentiation. Facioscapulohumeral muscular dystrophy has been genetically linked to reduced
numbers (≤ 8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4
polyadenylation signal haplotype. However, we have recently reported that 1.3%
of healthy individuals carry this molecular signature and 19% of subjects
affected by facioscapulohumeral muscular dystrophy do not carry alleles with
eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at
risk of carrying D4Z4 reduced alleles has become more complicated. To test for
additional prognostic factors, we measured the degree of motor impairment in a
large group of patients affected by facioscapulohumeral muscular dystrophy and
their relatives who are carrying D4Z4 reduced alleles. The clinical expression
of motor impairment was assessed in 530 subjects, 163 probands and 367
relatives, from 176 unrelated families according to a standardized clinical
score. The associations between clinical severity and size of D4Z4 allele,
degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2%
of relatives did not display any muscle functional impairment. This phenotype
was influenced by the degree of relation with proband, because 47.1% of second-
through fifth-degree relatives were unaffected, whereas only 27.5% of
first-degree family members did not show motor impairment. The estimated risk of
developing motor impairment by age 50 for relatives carrying a D4Z4 reduced
allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male
relatives had a mean score significantly higher than females (5.4 versus 4.0, P
= 0.003). No 4q haplotype was exclusively associated with the presence of
disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate,
the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one
generation. In conclusion, this large-scale analysis provides further
information that should be taken into account when counselling families in which
a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced
expression of disease observed in distant relatives suggests that a family's
genetic background plays a role in the occurrence of facioscapulohumeral
muscular dystrophy. These results indicate that the identification of new
susceptibility factors for this disease will require an accurate classification
of families. Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction
of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. The residual
number of D4Z4 units inversely correlates with clinical severity, but
significant clinical variability exists. Each unit contains a copy of the DUX4
retrogene. Repeat contractions are associated with changes in D4Z4 chromatin
structure that increase the likelihood of DUX4 expression in skeletal muscle,
but only when the repeat resides in a genetic background that contains a DUX4
polyadenylation signal. Mutations in the structural maintece of chromosomes
flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier
of D4Z4, also result in the increased likelihood of DUX4 expression in
individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to
D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may
act as a genetic modifier in FSHD1. We describe three unrelated individuals with
FSHD1 presenting an unusual high clinical severity based on their upper-sized
FSHD1 repeat array of nine units. Each of these individuals also carries a
mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the
SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the
SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4
expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We
conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which
the FSHD2 gene can act as modifier for disease severity in families affected by
FSHD1. |
Which G protein is essential in the formation and function of lamellipodia? | Recruitment of the small G-protein Rac1 to the plasma membrane is essential in inducing the local formation of specialized cellular processes termed lamellipodia. | EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor
(GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived
growth factor (PDGF), which stimulates sphingosine kinase and increases
intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or
inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also
activation of the small guanosine triphosphatase Rac, which is essential for
protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1,
as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our
results reveal a role for receptor cross-communication in which activation of a
GPCR by a receptor tyrosine kinase is critical for cell motility. EDG-1, encoded by the endothelial differentiation gene-1, is a heterotrimeric
guanine nucleotide binding protein-coupled receptor (GPCR) for
sphingosine-1-phosphate (SPP) that has been shown to stimulate angiogenesis and
cell migration in cultured endothelial cells. Unexpectedly, EDG-1 knockout
embryos had a normal blood vessel network, vasculogenesis and angiogenesis, but
died in utero owing to massive haemorrhaging as a result of failure of smooth
muscle cells and pericytes to migrate around the circumference and reinforce
endothelial tubes [Liu, Wada, Yamashita, Mi, Deng, Hobson, Rosenfeldt, Nava,
Chae, Lee, et al. (2000) J. Clin. Invest. 106, 951-961]. This vascular
maturation defect is similar to the phenotype of mice homozygous for disrupted
alleles of platelet-derived growth factor B-subunit homodimer (PDGF-BB) or its
receptor PDGFR-beta. We found that fibroblasts from EDG-1 null embryos did not
migrate toward PDGF or SPP, and inhibition of motility correlated with defective
activation of the small guanosine triphosphatase Rac, which is required for
lamellipodia formation and directional locomotion [Hobson, Rosenfeldt, Barak,
Olivera, Poulton, Caron, Milstien, and Spiegel (2001) Science 291, 1800-1803].
Moreover, we showed that PDGF-directed cell migration requires both sphingosine
kinase activation and expression of EDG-1, suggesting a functional link between
PDGF signalling and EDG-1. Indeed, treatment of wild-type cells with PDGF
transactivated EDG-1 as determined by translocation of beta-arrestin and
phosphorylation of EDG-1. These findings reveal a new paradigm for receptor
cross-communication in which activation of a GPCR by a receptor tyrosine kinase
is critical for cell motility. Our observations might also clarify the role of
EDG-1 in vascular maturation and angiogenesis. Protein-protein interactions are essential for many cellular processes. We have
developed a technology called light-activated dimerization (LAD) to artificially
induce protein hetero- and homodimerization in live cells using light. Using the
FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated
protein tags whose interaction is controlled by blue light. We demonstrated the
utility of this system with LAD constructs that can recruit the small G-protein
Rac1 to the plasma membrane and induce the local formation of lamellipodia in
response to focal illumination. We also generated a light-activated
transcription factor by fusing domains of GI and FKF1 to the DNA binding domain
of Gal4 and the transactivation domain of VP16, respectively, showing that this
technology is easily adapted to other systems. These studies set the stage for
the development of light-regulated signaling molecules for controlling receptor
activation, synapse formation and other signaling events in organisms. OBJECTIVE: Recent evidence suggests G-protein-coupled receptor-2-interacting
protein-1 (GIT1) overexpression in several human metastatic tumors, including
breast, lung, and prostate. Tumor metastasis is associated with an increase in
angiogenesis. We have showed previously that GIT1 is required for postnatal
angiogenesis during lung development. However, the functional role of GIT1 in
pathological angiogenesis during tumor growth is unknown.
APPROACH AND RESULTS: In the present study, we show inhibition of angiogenesis
in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor
growth in GIT1-knockout mice. We demonstrate that this is a result of impaired
directional migration of GIT1-depleted endothelial cells toward a vascular
endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in
the leading edge is critical for directional migration. We observed a
significant reduction in cortactin localization and lamellipodia formation in
the leading edge of GIT1-depleted endothelial cells. We specifically identified
that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin
complex localization to the leading edge. The mechanisms involved extracellular
signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and
activation of Rac1/Cdc42. Finally, using gain of function studies, we show that
a constitutively active mutant of cortactin restored directional migration of
GIT1-depleted cells.
CONCLUSION: Our data demonstrated that a GIT1-cortactin association through
GIT1-Spa homology domain is required for cortactin localization to the leading
edge and is essential for endothelial cell directional migration and tumor
angiogenesis. |
Which are the main functions of the APOBEC3 family of proteins? | The APOBEC3 family of cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1), and endogenous transposable elements, such as LINE-1 and Alu retrotransposons. | In the human genome the apolipoprotein B mRNA-editing enzyme catalytic
polypeptide (APOBEC)3 gene has expanded into a tandem array of genes termed
APOBEC3A-G. Two members of this family, APOBEC3G and APOBEC3F, have been found
to have potent activity against virion infectivity factor deficient (Deltavif)
human immunodeficiency virus 1 (HIV-1). These enzymes become encapsidated in
Deltavif HIV-1 virions and in the next round of infection deaminate the newly
synthesized reverse transcripts. The lentiviral Vif protein prevents the
deamination by inducing the degradation of APOBEC3G and APOBEC3F. We report here
that two additional APOBEC3 family members, APOBEC3B and APOBEC3C, have potent
antiviral activity against simian immuno-deficiency virus (SIV), but not HIV-1.
Both enzymes were encapsidated in HIV-1 and SIV virions and were active against
Deltavif SIV(mac) and SIV(agm). SIV Vif neutralized the antiviral activity of
APOBEC3C, but not that of APOBEC3B. APOBEC3B induced abundant G --> A mutations
in both wild-type and Deltavif SIV reverse transcripts. APOBEC3C induced
substantially fewer mutations. APOBEC3F was found to be active against SIV and
sensitive to SIV(mac) Vif. These findings raise the possibility that the
different APOBEC3 family members function to neutralize specific lentiviruses. Foamy viruses are a family of complex retroviruses that establish common,
productive infections in a wide range of nonhuman primates. In contrast, humans
appear nonpermissive for foamy virus replication, although zoonotic infections
do occur. Here we have analyzed the ability of primate and mouse APOBEC3G
proteins to inhibit the infectivity of primate foamy virus (PFV) virions
produced in their presence. We demonstrate that several APOBEC3 proteins can
potently inhibit the infectivity of a PFV-based viral vector. This inhibition
correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions,
due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A
hypermutation of PFV reverse transcripts. While inhibition of PFV virion
infectivity by primate APOBEC3 proteins was largely relieved by coexpression of
the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain
function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet
bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this
binding correlated with the specific inhibition of their incorporation into PFV
virions. Of note, both PFV Bet and a second Bet protein, derived from an African
green monkey foamy virus, rescued the infectivity of Vif-deficient human
immunodeficiency virus type 1 (HIV-1) virions produced in the presence of
African green monkey APOBEC3G and blocked the incorporation of this host factor
into HIV-1 virion particles. However, neither foamy virus Bet protein reduced
APOBEC3 protein expression levels in virion producer cells. While these data
identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif
auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein
function by distinct mechanisms. APOBEC3G is a potent antiretroviral factor, which belongs to the APOBEC
superfamily of cytidine deaminases. It deaminates cytidine to uridine in nascent
minus-strand viral DNA, inducing G-to-A hypermutation in the plus-strand viral
DNA. HIV-1 Vif protein overcomes the antiviral activity of APOBEC3G by targeting
it for ubiquitin-dependent degradation. Recent accumulating evidences that other
members of APOBEC proteins also show antiviral activity on a wide variety of
viruses suggest that APOBEC family proteins play a crucial role in an antiviral
defense as an innate immunity. Here, we review recent progress in research on
APOBEC3 proteins. Approximately 17% of the human genome is comprised of long interspersed nuclear
element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known
to be the cause of several genetic diseases, such as hemophilia A, Duchene
muscular dystrophy, and so on. The L1 retroelements are also able to cause colon
cancer, suggesting that L1 transposition could occur not only in germ cells, but
also in somatic cells if innate immunity would not function appropriately. The
mechanisms of L1 transposition restriction in the normal cells, however, are not
fully defined. We here show that antiretroviral innate proteins, human APOBEC3
(hA3) family members, from hA3A to hA3H, differentially reduce the level of L1
retrotransposition that does not correlate either with antiviral activity
against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of
subcellular localization. Importantly, hA3G protein inhibits L1
retrotransposition, in striking contrast to the recent reports. Inhibitory
effect of hA3 family members on L1 transposition might not be due to deaminase
activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins
act to differentially suppress uncontrolled transposition of L1 elements. Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for
viral spread and as a viral reservoir. Yet, cells of this lineage generally
resist HIV-1 infection when compared to cells of other lineages, a phenomenon
particularly acute during the early phases of infection. Here, we explore the
role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that
is highly expressed in myeloid cells, but so far lacks a known antiviral effect
against retroviruses. Using ectopic expression of APOBEC3A in established cell
lines and specific silencing in primary macrophages and dendritic cells, we
demonstrate that the pool of APOBEC3A in target cells inhibits the early phases
of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1,
specifically in cells of myeloid origins. In these cells, APOBEC3A affects the
amount of vDNA synthesized over the course of infection. The susceptibility to
the antiviral effect of APOBEC3A is conserved among primate lentiviruses,
although the viral protein Vpx coded by members of the SIV(SM)/HIV-2 lineage
provides partial protection from APOBEC3A during infection. Our results indicate
that APOBEC3A is a previously unrecognized antiviral factor that targets primate
lentiviruses specifically in myeloid cells and that acts during the early phases
of infection directly in target cells. The findings presented here open up new
venues on the role of APOBEC3A during HIV infection and pathogenesis, on the
role of the cellular context in the regulation of the antiviral activities of
members of the APOBEC3 family and more generally on the natural functions of
APOBEC3A. Human APOBEC3 (A3) proteins are cellular cytidine deaminases that potently
restrict the replication of retroviruses by hypermutating viral cDNA and/or
inhibiting reverse transcription. There are seven members of this family
including A3A, B, C, DE, F, G, and H, all encoded in a tandem array on human
chromosome 22. A3F and A3G are the most potent inhibitors of HIV-1, but only in
the absence of the virus-encoded protein, Vif. HIV-1 utilizes Vif to abrogate A3
functions in the producer cells. More specifically, Vif, serving as a substrate
receptor, facilitates ubiquitination of A3 proteins by forming a Cullin5
(Cul5)-based E3 ubiquitin ligase complex, which targets A3 proteins for rapid
proteasomal degradation. The specificity of A3 degradation is determined by the
ability of Vif to bind to the target. Several lines of evidence have suggested
that three distinct regions of A3 proteins are involved in the interaction with
Vif. Here, we review the biological functions of A3 family members with special
focus on A3G and base our analysis on the available structural information. Since the identification of APOBEC3G (A3G) as a potent restriction factor of
HIV-1, a tremendous amount of effort has led to a broadened understanding of
both A3G and the APOBEC3 (A3) family to which it belongs. In spite of the
fine-tuned viral counterattack to A3 activity, in the form of the HIV-1 Vif
protein, enthusiasm for leveraging the Vif : A3G axis as a point of clinical
intervention remains high. In an impressive explosion of information over the
last decade, additional A3 family members have been identified as antiviral
proteins, mechanistic details of the restrictive capacity of these proteins have
been elucidated, structure-function studies have revealed important molecular
details of the Vif : A3G interaction, and clinical cohorts have been scrutinized
for correlations between A3 expression and function and viral pathogenesis. In
the last year, novel and unexpected findings regarding the role of A3G in
immunity have refocused efforts on exploring the potential of harnessing the
natural power of this immune defense. These most recent reports allude to
functions of the A3 proteins that extend beyond their well-characterized
designation as restriction factors. The emerging story implicates the A3 family
as not only defense proteins, but also as participants in the broader innate
immune response. |
Which histone modifications are associated with constitutive heterochromatin? | Strong methylation at H3 lysine 9 occurred preferentially in heterochromatic chromocenters of Arabidopsis nuclei. In general, heterochromatin has been linked to trimethylation of H3 at lysine 9 and parsimony analysis reveal that histone H3K9 methylation is, next to histone deacetylation, the evolutionary most stable heterochromatic mark. Classical histone modifications associated with heterochromatin also include H3K27me1 and H3K27me2. H3K36me3 function is not restricted to actively transcribed regions only and may contribute to the composition of heterochromatin. Other histone methylation marks usually found in constitutive heterochromatin are H4K20me3, H3K9me3, and H3K79me3. H3S10P is a good marker of pericentromeric heterochromatin. | Histone methylation was first described more than 35 years ago, but its role has
remained enigmatic. Proposed functions range from transcriptional regulation to
the higher-order packaging of chromatin in preparation for mitotic condensation.
Histone methylation can occur on Arg or Lys residues, with an exquisite site
selectivity for Lys methylation at specific positions in the N-termini of
histones H3 and H4. Thus, Lys methylation joins acetylation and phosphorylation
as a third component of a 'histone code' that modifies the underlying chromatin
structure of the genetic information. Notably, in contrast to acetylation and
phosphorylation, Lys methylation appears to be a relatively stable histone
modification, thereby providing an ideal epigenetic mark for more long-term
maintece of chromatin states. The recent discovery of the first histone Lys
methyltransferase has allowed the identification of a molecular mechanism in
which the specific methylation of histone H3 at Lys9 generates a binding site
for heterochromatin-associated proteins. These findings have broad implications
for the overall functional organization of chromosome structure at constitutive
heterochromatin (e.g. centromeres) and for chromatin-dependent inheritance of
gene expression patterns. This review discusses how understanding this
methylation system should address some of the long-standing mysteries of
heterochromatin. Post-translational modification of histone tails is thought to modulate
higher-order chromatin structure. Combinations of modifications including
acetylation, phosphorylation and methylation have been proposed to provide marks
recognized by specific proteins. This is exemplified, in both mammalian cells
and fission yeast, by transcriptionally silent constitutive pericentric
heterochromatin. Such heterochromatin contains histones that are generally
hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of
histone H3 (H3-K9). Each of these modification states has been implicated in the
maintece of HP1 protein-binding at pericentric heterochromatin, in
transcriptional silencing and in centromere function. In particular, H3-K9
methylation is thought to provide a marking system for the establishment and
maintece of stably repressed regions and heterochromatin subdomains. To
address the question of how these two types of modifications, as well as other
unidentified parameters, function to maintain pericentric heterochromatin, we
used a combination of histone deacetylase inhibitors, RNAse treatments and an
antibody raised against methylated branched H3-K9 peptides. Our results show
that both H3-K9 acetylation and methylation can occur on independent sets of H3
molecules in pericentric heterochromatin. In addition, we identify an RNA- and
histone modification-dependent structure that brings methylated H3-K9 tails
together in a specific configuration required for the accumulation of HP1
proteins in these domains. Facioscapulohumeral muscular dystrophy (FSHD) is a unique domit disorder
involving shortening of an array of tandem 3.3 kb repeats. This copy-number
polymorphic repeat, D4Z4, is present in arrays at both 4q35 and 10q26, but only
4q35 arrays with one to 10 copies of the repeat are linked to FSHD. The most
popular model for how the 4q35 array-shortening causes FSHD is that it results
in a loss of postulated D4Z4 heterochromatinization, which spreads proximally,
leading to overexpression of FSHD genes in cis. This would be similar to a loss
of position-effect variegation (PEV) in Drosophila. To test for the putative
heterochromatinization, we quantitated chromatin immunoprecipitation with an
antibody for acetylated histone H4 that discriminates between constitutive
heterochromatin and unexpressed euchromatin. Contrary to the above model, H4
acetylation levels of a non-repeated region adjacent to the 4q35 and 10q26 D4Z4
arrays in normal and FSHD lymphoid cells were like those in unexpressed
euchromatin and not constitutive heterochromatin. Also, these control and FSHD
cells displayed similar H4 hyperacetylation (like that of expressed genes) at
the 5' regions of 4q35 candidate genes FRG1 and ANT1. Contrary to the
loss-of-PEV model and a recent report, there was no position-dependent increase
in transcript levels from these genes in FSHD skeletal muscle samples compared
with controls. Our results favor a new model for the molecular genetic etiology
of FSHD, such as, differential long-distance cis looping that depends upon the
presence of a 4q35 D4Z4 array with less than a threshold number of copies of the
3.3 kb repeat. Histone modifications are implicated in regulating chromatin condensation but it
is unclear how they differ between constitutive heterochromatin and unexpressed
euchromatin. Chromatin immunoprecipitation (ChIP) assays were done on various
human cell populations using antibodies specific for acetylated or methylated
forms of histone H3 or H4. Analysis of the immunoprecipitates was by
quantitative real-time PCR or semi-quantitative PCR (SQ-PCR). Of eight tested
antibodies, the one for histone H4 acetylated at lysine 4, 8, 12, or 16 was best
for distinguishing constitutive heterochromatin from unexpressed euchromatin,
but differences in the extent of immunoprecipitation of these two types of
chromatin were only modest, although highly reproducible. With this antibody,
there was an average of 2.5-fold less immunoprecipitation of three constitutive
heterochromatin regions than of four unexpressed euchromatic gene regions and
about 15-fold less immunoprecipitation of these heterochromatin standards than
of two constitutively expressed gene standards (P <0.001). We also analyzed
histone acetylation and methylation by immunocytochemistry with antibodies to H4
acetylated at lysine 8, H3 trimethylated at lysine 9, and H3 methylated at
lysine 4. In addition, immunocytochemical analysis was done with an antibody to
heterochromatin protein 1alpha (HP1alpha), whose preferential binding to
heterochromatin has been linked to trimethylation of H3 at lysine 9. Our
combined ChIP and immunocytochemical results suggest that factors other than
hypoacetylation of the N-terminal tails of H4 and hypermethylation of H3 at
lysine 9 can play an important role in determining whether a chromatin sequence
in mammalian cells is constitutively heterochromatic. Posttranslational histone modifications and histone variants form a unique
epigenetic landscape on mammalian chromosomes where the principal epigenetic
heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are
inversely localized in relation to each other. Trimethylated H3(K9) marks
pericentromeric constitutive heterochromatin and the male Y chromosome, while
H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a
lysosomal and nuclear protease, cathepsin L, causes a global redistribution of
epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated
H3(K9) decrease dramatically, concomitant with its relocation away from
heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin
and the Y chromosome. This change is also associated with global relocation of
heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from
constitutive heterochromatin; however, it does not affect DNA methylation or
chromosome segregation, phenotypes commonly associated with impaired histone
H3(K9) methylation. Therefore, the key constitutive heterochromatin determits
can dynamically redistribute depending on physiological context but still
maintain the essential function(s) of chromosomes. Thus, our data show that
cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric
heterochromatin and the Y chromosome through a novel mechanism that does not
involve DNA methylation or affect heterochromatin structure and operates on both
somatic and sex chromosomes. In eukaryotes, histone methylation is an epigenetic mechanism associated with a
variety of functions related to gene regulation or genomic stability. Recently
analyzed H3K9 methyltransferases (HMTases) as SUV39H1, Clr4p, DIM-5, Su(var)3-9
or SUVH2 are responsible for the establishment of histone H3 lysine 9
methylation (H3K9me), which is intimately connected with heterochromatinization.
In this review, available data will be evaluated concerning (1) the phylogenetic
distribution of H3K9me as heterochromatin-specific histone modification and its
evolutionary stability in relation to other epigenetic marks, (2) known families
of H3K9 methyltransferases, (3) their responsibility for the formation of
constitutive heterochromatin and (4) the evolution of Su(var)3-9-like and
SUVH-like H3K9 methyltransferases. Compilation and parsimony analysis reveal
that histone H3K9 methylation is, next to histone deacetylation, the
evolutionary most stable heterochromatic mark, which is established by at least
two subfamilies of specialized heterochromatic HMTases in almost all studied
eukaryotes. In contrast to stably repressive, constitutive heterochromatin and stably
active, euchromatin, facultative heterochromatin has the capacity to alternate
between repressive and activated states of transcription. As such, it is an
instructive source to understand the molecular basis for changes in chromatin
structure that correlate with transcriptional status. Sirtuin 1 (SIRT1) and
suppressor of variegation 3-9 homologue 1 (SUV39H1) are amongst the enzymes
responsible for chromatin modulations associated with facultative
heterochromatin formation. SUV39H1 is the principal enzyme responsible for the
accumulation of histone H3 containing a tri-methyl group at its lysine 9
position (H3K9me3) in regions of heterochromatin. SIRT1 is an NAD+-dependent
deacetylase that targets histone H4 at lysine 16 (refs 3 and 4), and through an
unknown mechanism facilitates increased levels of H3K9me3 (ref. 3). Here we show
that the mammalian histone methyltransferase SUV39H1 is itself targeted by the
histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation
at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly
with, recruits and deacetylates SUV39H1, and these activities independently
contribute to elevated levels of SUV39H1 activity resulting in increased levels
of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent
H3K9me3 and impairs localization of heterochromatin protein 1. These findings
demonstrate a functional link between the heterochromatin-related histone
methyltransferase SUV39H1 and the histone deacetylase SIRT1. In mammals, DNA methylation is an important epigenetic mark that is associated
with gene silencing, particularly in constitutive heterochromatin. However, the
effect of DNA methylation on other epigenetic properties of chromatin is
controversial. In this study, we show that inhibition of DNA methylation in
mouse fibroblast cells affects histone modification and the subnuclear
localization of histone H3.3 in a cell cycle-dependent manner. Using a DNA
methyltransferase (Dnmt) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), we found
that reduced levels of DNA methylation were associated with the activation of
transcription from centromeric and pericentromeric satellite repeats. The
de-repressed pericentromeric chromatin was enriched in euchromatic histone
modifications such as acetylation of histone H4, and di- and tri-methylation of
lysine 4 on histone H3. Spatio-temporal analysis showed that the accumulation of
these euchromatic histone modifications occurred during the second S phase
following 5-aza-dC treatment, corresponding precisely with a shift in
replication timing of the pericentromeric satellite repeats from middle/late S
phase to early S phase. Moreover, we found that histone H3.3 was deposited on
the pericentromeric heterochromatin prior to the accumulation of the euchromatic
histone modifications. These results suggest that DNA CpG methylation is
essential for the proper organization of pericentromeric heterochromatin in
differentiated mouse cells. H3 lysine 9 trimethylation (H3K9me3) is a histone posttranslational modification
(PTM) that has emerged as hallmark of pericentromeric heterochromatin. This
constitutive chromatin domain is composed of repetitive DNA elements, whose
transcription is differentially regulated. Mammalian cells contain three HP1
proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are
thought to mediate the effects of this histone PTM. However, the mechanisms of
HP1 chromatin regulation and the exact functional role at pericentromeric
heterochromatin are still unclear. Here, we identify activity-dependent
neuroprotective protein (ADNP) as an H3K9me3 associated factor. We show that
ADNP does not bind H3K9me3 directly, but that interaction is mediated by all
three HP1 isoforms in vitro. However, in cells ADNP localization to areas of
pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a
PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain
involved in HP1 dependent H3K9me3 association and localization to
pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1
distribution and heterochromatic histone and DNA modifications, we found ADNP
silencing major satellite repeats. Our results identify a novel factor in the
translation of H3K9me3 at pericentromeric heterochromatin that regulates
transcription. The mammalian genome contains numerous regions known as facultative
heterochromatin, which contribute to transcriptional silencing during
development and cell differentiation. We have analyzed the pattern of histone
modifications associated with facultative heterochromatin within the mouse
imprinted Snurf-Snrpn cluster, which is homologous to the human Prader-Willi
syndrome genomic region. We show here that the maternally inherited Snurf-Snrpn
3-Mb region, which is silenced by a potent transcription repressive mechanism,
is uniformly enriched in histone methylation marks usually found in constitutive
heterochromatin, such as H4K20me3, H3K9me3, and H3K79me3. Strikingly, we found
that trimethylated histone H3 at lysine 36 (H3K36me3), which was previously
identified as a hallmark of actively transcribed regions, is deposited onto the
silenced, maternally contributed 3-Mb imprinted region. We show that H3K36me3
deposition within this large heterochromatin domain does not correlate with
transcription events, suggesting the existence of an alternative pathway for the
deposition of this histone modification. In addition, we demonstrate that
H3K36me3 is markedly enriched at the level of pericentromeric heterochromatin in
mouse embryonic stem cells and fibroblasts. This result indicates that H3K36me3
is associated with both facultative and constitutive heterochromatin. Our data
suggest that H3K36me3 function is not restricted to actively transcribed regions
only and may contribute to the composition of heterochromatin, in combination
with other histone modifications. Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of
cellular processes, such as chromosome condensation and gene
activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates
in the pericentromeric heterochromatin during the late G2 phase, and
phosphorylation spreads throughout the chromosomes arms in prophase, being
maintained until the onset of anaphase when it gets dephosphorylated.
Considerable studies have been carried out about H3S10P in different organisms;
however, there is little information about this histone modification in
mammalian embryos. We hypothesized that this epigenetic modification could also
be a marker of pericentromeric heterochromatin in preimplantation embryos. We
therefore followed the H3S10P distribution pattern in the G1/S and G2 phases
through the entire preimplantation development in in vivo mouse embryos. We paid
special attention to its localization relative to another pericentromeric
heterochromatin marker, HP1β and performed immunoFISH using specific
pericentromeric heterochromatin probes. Our results indicate that H3S10P
presents a remarkable distribution pattern in preimplantation mouse embryos
until the 4-cell stage and is a better marker of pericentromeric heterochromatin
than HP1β. After the 8-cell stage, H3S10P kinetic is more similar to the somatic
one, initiating during G2 in chromocenters and disappearing upon telophase.
Based on these findings, we believe that H3S10P is a good marker of
pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as
it labels both parental genomes and that it can be used to further investigate
epigenetic regulation and heterochromatin mechanisms in early preimplantation
embryos. |
What is the main application of SWATH-MS in proteomics? | Using the method called SWATH-MS one might ask sample sets for the presence and quantity of essentially any protein of interest. | The prevalence of metabolic disorders (MDs), especially diabetes, is rapidly
increasing worldwide, leading to an increasing risk of cardiovascular and other
socially relevant complications. To boost MD biomarker discovery, advanced
proteomics can harmonize metabolomics. Indeed, the rapid development of mass
spectrometry (MS) has designated proteomics as an emerging platform to
interrogate the plasma/serum proteome for the discovery of next-generation
biomarkers exploitable for risk assessment, early detection and prognosis of
MDs. Preanalytical plasma/serum treatment, such as combinatorial peptide ligand
libraries with o-liquid chromatography coupled with tandem MS or selected
reaction monitoring coupled to triple-quadrupole time-of-flight instruments, are
proven clinical laboratory techniques for quantitative analyses. New strategies,
such as SWATH™ MS, which allows us to systematically characterize and quantify
query sample sets of 'any protein of interest' in complex biological samples,
may dramatically improve next-generation MD biomarkers, especially considering
the plethora of candidates coming from the 'bioreactor' gut microbiota affecting
MD onset and progression. Candida albicans public proteomic datasets, though growing steadily in the last
few years, still have a very limited presence in online repositories. We report
here the creation of a C. albicans PeptideAtlas comprising near 22,000 distinct
peptides at a 0.24% False Discovery Rate (FDR) that account for over 2500
canonical proteins at a 1.2% FDR. Based on data from 16 experiments, we attained
coverage of 41% of the C. albicans open reading frame sequences (ORFs) in the
database used for the searches. This PeptideAtlas provides several useful
features, including comprehensive protein and peptide-centered search
capabilities and visualization tools that establish a solid basis for the study
of basic biological mechanisms key to virulence and pathogenesis such as
dimorphism, adherence, and apoptosis. Further, it is a valuable resource for the
selection of candidate proteotypic peptides for targeted proteomic experiments
via Selected Reaction Monitoring (SRM) or SWATH-MS.
BIOLOGICAL SIGNIFICANCE: This C. albicans PeptideAtlas resolves the previous
absence of fungal pathogens in the PeptideAtlas project. It represents the most
extensive characterization of the proteome of this fungus that exists up to the
current date, including evidence for uncharacterized ORFs. Through its web
interface, PeptideAtlas supports the study of interesting proteins related to
basic biological mechanisms key to virulence such as apoptosis, dimorphism and
adherence. It also provides a valuable resource to select candidate proteotypic
peptides for future (SRM) targeted proteomic experiments. This article is part
of a Special Issue entitled: Trends in Microbial Proteomics. |
Do selenoproteins and selenium play a role in prostate cancer prevention? | No, although initial epidemiological studies on humans and on animal and cell- based models indicated that selenoproteins may be protecting against prostate cancer, more research is needed to improve the understanding of selenium metabolism and requirements for optimal health. | Prostate cancer (PC) is the most common cancer in men and a leading cause of
cancer death. Prostatic gland accumulates reasonably high amount of selenium
(Se), the element that prevents the development of PC. It is hypothesized that
some selenoproteins inhibit the transformation of normal prostate epithelium
into neoplasm. We studied Se levels in whole blood, plasma and prostate of 32 PC
and 40 benign prostate hyperplasia (BPH) patients and in the control group
composed of 39 healthy subjects. The selenoenzyme glutathione peroxidase
(GSH-Px) was also measured in the patients' red cells, plasma and prostate
tissue. Se concentration in whole blood and plasma in both groups of patients
was lower as compared with controls, while in prostate gland it was
significantly higher in PC than in BPH patients and controls. Red cell GSH-Px
activity was the same in PC patients and controls but significantly lower in BPH
patients. Plasma GSH-Px activity was significantly lower in PC patients than in
the control group, and prostate GSH-Px activity was significantly lower in PC
patients as compared with BPH patients. Since Se has anticancer properties, it
is very likely that its low level in blood may facilitate the development of
cancer. A higher level of Se in prostate of PC patients has no influence on
GSH-Px activity in the gland. Considerable animal and human data have indicated that selenium is effective in
reducing the incidence of several different types of cancer, including that of
the prostate. However, the mechanism by which selenium inhibits carcinogenesis
remains unknown. One possibility is that dietary selenium influences the levels
of selenium-containing proteins, or selenoproteins. Selenoproteins contain
selenium in the form of selenocysteine and perform a variety of cellular
functions, including antioxidant defense. To determine whether the levels of
selenoproteins can influence carcinogenesis independent of selenium intake, a
unique mouse model was developed by breeding two transgenic animals: mice with
reduced selenoprotein levels because of the expression of an altered
selenocysteine-tRNA (i6A-) and mice that develop prostate cancer because of the
targeted expression of the SV40 large T and small t oncogenes to that organ
[C3(1)/Tag]. The resulting bigenic animals (i6A-/Tag) and control WT/Tag mice
were assessed for the presence, degree, and progression of prostatic epithelial
hyperplasia and nuclear atypia. The selenoprotein-deficient mice exhibited
accelerated development of lesions associated with prostate cancer progression,
implicating selenoproteins in cancer risk and development and raising the
possibility that selenium prevents cancer by modulating the levels of these
selenoproteins. Selenium may affect prostate cancer risk via its plasma carrier selenoprotein P
which shows dramatically reduced expression in prostate cancer tumors and cell
lines. The selenoprotein P (SEPP1) Ala234 single nucleotide polymorphism (SNP)
allele is associated with lower plasma selenoprotein P in men, reducing the
concentration/activity of other antioxidant selenoproteins. Selenium status also
modifies the effect of the mitochondrial superoxide dismutase (SOD2) SNP
Ala16Val on prostate cancer risk. We investigated the relationship of these SNPs
with prostate cancer risk. DNA from 2,975 cases and 1,896 age-matched controls
from the population-based Prostate Cancer in Sweden study were genotyped using
TaqMan assays. Cases were designated aggressive or nonaggressive prostate
cancers at diagnosis by clinical criteria. Association with prostate cancer was
investigated by logistic regression; gene-gene interaction using a general
linear model. The mean plasma selenium concentration measured in 169 controls
was relatively low (76.0 +/- 17.2 microg/L). SNP genotype distributions were in
Hardy-Weinberg equilibrium. SOD2-Ala16+ men were at a greater risk of prostate
cancer [odds ratios (OR), 1.19; 95% confidence intervals (CI), 1.03-1.37]
compared with SOD2-Val16 homozygotes. Men homozygous for SEPP1-Ala234 who were
also SOD2-Ala16+ had a higher risk of prostate cancer (OR, 1.43; 95% CI,
1.17-1.76) and aggressive prostate cancer (OR, 1.60; 95% CI, 1.22-2.09) than
those who were SOD2-Val16 homozygotes (interaction, prostate cancer P = 0.05;
aggressive prostate cancer P = 0.01). This interaction was stronger in
ever-smokers: SOD2-Ala16+ men homozygous for SEPP1-Ala234 had an almost doubled
risk of prostate cancer (OR, 1.97; 95% CI, 1.33-2.91; interaction P = 0.001). In
a low-selenium population, SOD2-Ala16+ men homozygous for SEPP1-Ala234 are at an
increased risk of prostate cancer/aggressive prostate cancer especially if
ever-smokers, because they are likely to produce more mitochondrial H(2)O(2)
that they cannot remove, thereby promoting prostate tumor cell proliferation and
migration. The recently completed Selenium and Vitamin E Cancer Prevention Trial (SELECT)
was one of the largest human cancer prevention trials ever undertaken. Its
purpose was to assess the role of selenium and vitamin E in prostate cancer
prevention, but SELECT found no decline in prostate cancer. Comparison of this
study to other clinical trials involving selenium and to the results of animal
studies suggests that the source of the selenium supplement, L-selenomethionine,
and the relatively high initial levels of selenium in the enrolled men may have
contributed to this outcome. Further analysis of the clinical and animal data
highlights the need for mechanistic studies to better understand selenium
biology in order to target dietary selenium to appropriate subsets of the human
population: those individuals most likely to benefit from this micronutrient. Selenium (Se) is essentially needed for the biosynthesis of selenoproteins. Low
Se intake causes reduced selenoprotein biosynthesis and constitutes a risk
factor for tumorigenesis. Accordingly, some Se supplementation trials have
proven effective to reduce prostate cancer risk, especially in poorly supplied
individuals. Because Se metabolism is controlled by selenoprotein P (SEPP), we
have tested whether circulating SEPP concentrations correlate to prostate cancer
stage and grade. A total of 190 men with prostate cancer (n = 90) and "no
evidence of maligcy" (NEM; n = 100) histologically confirmed by prostate
biopsy were retrospectively analyzed for established tumor markers and for their
Se and SEPP status. Prostate specific antigen (PSA), free PSA, total Se, and
SEPP concentrations were determined from serum samples and compared with
clinicopathologic parameters. The diagnostic performance was analyzed with
receiver operating characteristic curves. Median Se and SEPP concentrations
differed significantly (P < 0.001) between the groups. Median serum Se
concentrations in the 25th to 75th percentile were 95.9 microg/L (82-117.9) in
NEM patients and 81.4 microg/L (67.9-98.4) in prostate cancer patients.
Corresponding serum SEPP concentrations were 3.4 mg/L (1.9-5.6) in NEM and 2.9
mg/L (1.1-5.5) in prostate cancer patients. The area under the curve (AUC) of a
marker combination with age, PSA, and percent free PSA (%fPSA) in combination
with the SEPP concentration, yielded the highest diagnostic value (AUC 0.80)
compared with the marker combination without SEPP (AUC 0.77) or %fPSA (AUC
0.76). We conclude that decreased SEPP concentration in serum might represent an
additional valuable marker for prostate cancer diagnostics. BACKGROUND: Evidence for an association between selenium status and prostate
cancer risk is still inconclusive. Anticarcinogenic effects of selenium are
supposedly mediated through cellular protective and redox properties of
selenoenzymes in vivo. We evaluated the association between serum selenium
status and prostate cancer risk in a population with relative low selenium
concentrations considering effect modification by genetic variants in
selenoprotein genes.
MATERIALS AND METHODS: A case-control study of 248 incident prostate cancer
cases and 492 matched controls was nested within the EPIC-Heidelberg cohort.
Baseline blood samples were analyzed for serum selenium and selenoprotein P
concentrations and glutathione peroxidase activity. Genotyping was carried out
for SEP15 (rs5859, rs540049), SEPP1 (rs3877899, rs7579), GPX1 (rs1050450), and
GPX4 (rs713041). Conditional logistic regression was used to calculate adjusted
odds ratios (OR) and 95% confidence intervals (95% CI).
RESULTS: The OR for prostate cancer was 0.89 (95% CI, 0.79-1.01) per 10 μg/L
increase of serum selenium concentration. This association was modified by
rs1050450 (C>T) in GPX1 (P(interaction) = 0.03), with carriers of one or two T
alleles having a significantly reduced OR of 0.87 (95% CI, 0.76-0.99).
Furthermore, there was an association between rs7579 genotype in SEPP1 and
prostate cancer risk (OR, 1.72; 95% CI, 0.99-2.98).
CONCLUSIONS: Our results support a role of selenium and polymorphisms in
selenoenzymes in prostate cancer etiology, which warrants confirmation in future
studies.
IMPACT: These findings might help to explain biological effects of selenium in
prostate cancer development in order to overcome inconsistencies arising from
former studies. BACKGROUND: Data from human epidemiological studies, cultured mammalian cells,
and animal models have supported a potentially beneficial role of selenium (Se)
in prostate cancer prevention. In addition, Se-containing proteins including
members of the glutathione peroxidase (GPx) family and Selenium-Binding Protein
1 (SBP1) have been linked to either cancer risk or development. For example,
SBP1 levels are typically reduced in tumors compared to non-cancerous tissue,
with the degree of reduction associated with increasingly poor clinical outcome.
METHODS: In order to investigate inter-relationships between blood and tissue Se
levels and GPx activity, tissue SBP1 levels, and disease aggressiveness using
the Gleason score, we measured levels of selenium and selected selenoproteins in
fasting serum and histologically normal prostate tissues obtained from 24 men
undergoing radical prostatectomy for the treatment of localized prostate cancer.
RESULTS: GPx enzyme activity was inversely correlated with SBP1 levels in
prostate tissue as determined by densitometry of Western blots obtained using
anti-SBP1 antibodies [partial Spearman's correlation coefficients and
corresponding P-values overall and in African-Americans = -0.42 (0.08) and -0.53
(0.10), respectively], which is consistent with previous observations in
cultured cells and mice. Of particular interest was the positive correlation
between tissue GPx activity and Gleason score, with this relationship achieving
statistical significance among African-Americans (r = 0.67, P = 0.02).
CONCLUSION: These studies support the continued investigation of the role of Se
and selenoproteins in prostate cancer prevention, development, and prognosis. Increased dietary intake of Selenium (Se) has been suggested to lower prostate
cancer mortality, but supplementation trials have produced conflicting results.
Se is incorporated into 25 selenoproteins. The aim of this work was to assess
whether risk of prostate cancer is affected by genetic variants in genes coding
for selenoproteins, either alone or in combination with Se status. 248 cases and
492 controls from an EPIC-Heidelberg nested case-control study were subjected to
two-stage genotyping with an initial screening phase in which 384 tagging-SNPs
covering 72 Se-related genes were determined in 94 cases and 94 controls using
the Illumina Goldengate methodology. This analysis was followed by a second
phase in which genotyping for candidate SNPs identified in the first phase was
carried out in the full study using Sequenom. Risk of high-grade or advanced
stage prostate cancer was modified by interactions between serum markers of Se
status and genotypes for rs9880056 in SELK, rs9605030 and rs9605031 in TXNRD2,
and rs7310505 in TXNRD1. No significant effects of SNPs on prostate cancer risk
were observed when grade or Se status was not taken into account. In conclusion,
the risk of high-grade or advanced-stage prostate cancer is significantly
altered by a combination of genotype for SNPs in selenoprotein genes and Se
status. The findings contribute to explaining the biological effects of selenium
intake and genetic factors in prostate cancer development and highlight
potential roles of thioredoxin reductases and selenoprotein K in tumour
progression. |
What can Nothobranchius furzeri be used as a model system for? | N. furzeri an interesting model system to investigate the effects of experimental manipulations on longevity and age-related pathologies.
N. furzeri could represent a model system for studying the genetic control of life-history traits in natural populations.
N. furzeri could be a very useful model for comparative genomics of aging.
It can be employed to test the effects of experimental manipulation on aging and apharmacological research. | Aging research in vertebrates is hampered by the lack of short-lived models.
Annual fishes of the genus Nothobranchius live in East African seasonal ponds.
Their life expectancy in the wild is limited by the duration of the wet season
and their lifespan in captivity is also short. Nothobranchius are popular
aquarium fishes and many different species are kept as captive strains,
providing rich material for comparative studies. The present paper aims at
reviving the interest in these fishes by reporting that: (1) Nothobranchius can
be cultured, and their eggs stored dry at room temperature for months or years,
offering inexpensive methods of embryo storage; (2) Nothobranchius show
accelerated growth and expression of aging biomarkers at the level of histology
and behaviour; (3) the species Nothobranchius furzeri has a maximum lifespan of
only 3 months and offers the possibility to perform investigations thus far
unthinkable in a vertebrate, such as drug screening with life-long
pharmacological treatments and experimental evolution; (4) when the lifespan of
different species is compared, a general correlation is found between wet season
duration in their natural habitat and longevity in captivity; and (5) vertebrate
aging-related genes, such as p66Shc and MTP, can be easily isolated in
Nothobranchius by homology cloning. These fishes can become excellent models for
aging studies. They can be employed to test the effects of experimental
manipulation on aging at a pace comparable with that of Drosophila and to probe
the effects of natural selection on the evolution of aging-related genes. The natural phytoalexin resveratrol, found in grapes and red wine, recently rose
to public fame for its positive effects on longevity in yeasts, worms and flies.
Resveratrol anti-cancer and anti-inflammatory in vitro action on mammalian cell
cultures also suggest a possible positive effect on human health and
life-expectancy. To study the effects of resveratrol on vertebrate aging is
obviously a particularly relevant question. We have studied resveratrol effects
in a very short-lived vertebrate: the annual fish Nothobranchius furzeri.
Resveratrol treatment prolonged lifespan and delayed the onset of age-related
dysfunctions in this fish. This result identifies resveratrol as the first
molecule which consistently retards aging in organisms as diverse as yeast,
worm, fly and fish, but it also reveals the potential of this short-lived fish
as an animal model for pharmacological research. Moreover, being related to
stickleback (Gasterosteus aculeatus) the "pufferfishes" Takifugu and Tetraodon,
and even more closely related to medaka (Oryzias latipes), it can greatly
beneficiate from the recent development of genomic resources for these fish
models and in the future become a complete model system for the aging research
community. Genetic and pharmacological research on aging is hampered by the lifespan of
available vertebrate models. We recently initiated studies on Nothobranchius
furzeri, a species with a maximum life expectancy in captivity of just three
months which represents the shortest documented captive lifespan for a
vertebrate. Further research on N. furzeri has demonstrated that 1. Short
lifespan is tied with explosive growth and accelerated sexual maturation. 2.
Short lifespan is correlated with expression of age-related behavioral and
histological changes. 3. Lifespan and expression of age-related markers can be
modulated by water temperature. 4. Resveratrol, a drug characterized for its
life-extending action in Caenorhabditis elegans and Drosophila, increases
lifespan and retards expression of age-related markers. 5. Aging-related genes
can be easily isolated by homology cloning. Finally, different populations or
species of Nothobranchius show large-scale differences in captive lifespan. In
the last three years, N. furzeri has moved from biological curiosity to a
promising model system for drug validation. Furthermore, this species occupies a
favorable position in the Teleost's "tree of life". It is very close to the
Japanese Medaka, and close to the pufferfishes and stickleback and might
represent a very useful model for comparative genomics of aging. BACKGROUND: A laboratory inbred strain of the annual fish Nothobranchius furzeri
shows exceptionally short life expectancy and accelerated expression of age
markers. In this study, we analyze new wild-derived lines of this short-lived
species.
METHODOLOGY/PRINCIPAL FINDINGS: We characterized captive survival and
age-related traits in F1 and F2 offspring of wild-caught N. furzeri.
Wild-derived N. furzeri lines showed expression of lipofuscin and
neurodegeneration at age 21 weeks. Median lifespan in the laboratory varied from
to 20 to 23 weeks and maximum lifespan from 25 to 32 weeks. These data
demonstrate that rapid age-dependent decline and short lifespan are natural
characteristics of this species. The N. furzeri distribution range overlaps with
gradients in altitude and aridity. Fish from more arid habitats are expected to
experience a shorter survival window in the wild. We tested whether captive
lines stemming from semi-arid and sub-humid habitats differ in longevity and
expression of age-related traits. We detected a clear difference in
age-dependent cognitive decline and a slight difference in lifespan (16% for
median, 15% for maximum lifespan) between these lines. Finally, we observed
shorter lifespan and accelerated expression of age-related markers in the inbred
laboratory strain compared to these wild-derived lines.
CONCLUSIONS/SIGNIFICANCE: Owing to large differences in aging phenotypes in
different lines, N. furzeri could represent a model system for studying the
genetic control of life-history traits in natural populations. The short-lived annual fish Nothobranchius furzeri shows extremely short captive
life span and accelerated expression of age markers, making it an interesting
model system to investigate the effects of experimental manipulations on
longevity and age-related pathologies. Here, we tested the effects of dietary
restriction (DR) on mortality and age-related markers in N. furzeri. DR was
induced by every other day feeding and the treatment was performed both in an
inbred laboratory line and a longer-lived wild-derived line. In the inbred
laboratory line, DR reduced age-related risk and prolonged maximum life span. In
the wild-derived line, DR induced early mortality, did not reduce general
age-related risk and caused a small but significant extension of maximum life
span. Analysis of age-dependent mortality revealed that DR reduced demographic
rate of aging, but increased baseline mortality in the wild-derived strain. In
both inbred- and wild-derived lines, DR prevented the expression of the age
markers lipofuscin in the liver and Fluoro-Jade B (neurodegeneration) in the
brain. DR also improved performance in a learning test based on conditioning
(active avoidance in a shuttle box). Finally, DR induced a paradoxical
up-regulation of glial fibrillary acidic protein in the brain. |
What is the function of TALENs? | These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. Artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. | Technology development has always been one of the forces driving breakthroughs
in biomedical research. Since the time of Thomas Morgan, Drosophilists have,
step by step, developed powerful genetic tools for manipulating and functionally
dissecting the Drosophila genome, but room for improving these technologies and
developing new techniques is still large, especially today as biologists start
to study systematically the functional genomics of different model organisms,
including humans, in a high-throughput manner. Here, we report, for the first
time in Drosophila, a rapid, easy, and highly specific method for modifying the
Drosophila genome at a very high efficiency by means of an improved
transcription activator-like effector nuclease (TALEN) strategy. We took
advantage of the very recently developed "unit assembly" strategy to assemble
two pairs of specific TALENs designed to modify the yellow gene (on the sex
chromosome) and a novel autosomal gene. The mRNAs of TALENs were subsequently
injected into Drosophila embryos. From 31.2% of the injected F(0) fertile flies,
we detected inheritable modification involving the yellow gene. The entire
process from construction of specific TALENs to detection of inheritable
modifications can be accomplished within one month. The potential applications
of this TALEN-mediated genome modification method in Drosophila are discussed. The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate
biology and human disease with a rich array of in vivo genetic and molecular
tools. However, the inability to readily modify the genome in a targeted fashion
has been a bottleneck in the field. Here we show that improvements in artificial
transcription activator-like effector nucleases (TALENs) provide a powerful new
approach for targeted zebrafish genome editing and functional genomic
applications. Using the GoldyTALEN modified scaffold and zebrafish delivery
system, we show that this enhanced TALEN toolkit has a high efficiency in
inducing locus-specific DNA breaks in somatic and germline tissues. At some
loci, this efficacy approaches 100%, including biallelic conversion in somatic
tissues that mimics phenotypes seen using morpholino-based targeted gene
knockdowns. With this updated TALEN system, we successfully used single-stranded
DNA oligonucleotides to precisely modify sequences at predefined locations in
the zebrafish genome through homology-directed repair, including the
introduction of a custom-designed EcoRV site and a modified loxP (mloxP)
sequence into somatic tissue in vivo. We further show successful germline
transmission of both EcoRV and mloxP engineered chromosomes. This combined
approach offers the potential to model genetic variation as well as to generate
targeted conditional alleles. Transcription activator-like effector nucleases (TALENs) are programmable
nucleases that join FokI endonuclease with the modular DNA-binding domain of
TALEs. Although zinc-finger nucleases enable a variety of genome modifications,
their application to genetic engineering of livestock has been slowed by
technical limitations of embryo-injection, culture of primary cells, and
difficulty in producing reliable reagents with a limited budget. In contrast, we
found that TALENs could easily be manufactured and that over half (23/36, 64%)
demonstrate high activity in primary cells. Cytoplasmic injections of TALEN
mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of
embryos analyzed, a portion of which harbored biallelic modification. We also
developed a simple transposon coselection strategy for TALEN-mediated gene
modification in primary fibroblasts that enabled both enrichment for modified
cells and efficient isolation of modified colonies. Coselection after treatment
with a single TALEN-pair enabled isolation of colonies with mono- and biallelic
modification in up to 54% and 17% of colonies, respectively. Coselection after
treatment with two TALEN-pairs directed against the same chromosome enabled the
isolation of colonies harboring large chromosomal deletions and inversions (10%
and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal
fibroblasts were effective nuclear donors for cloning, resulting in the creation
of miniature swine containing mono- and biallelic mutations of the LDL receptor
gene as models of familial hypercholesterolemia. TALENs thus appear to represent
a highly facile platform for the modification of livestock genomes for both
biomedical and agricultural applications. |
Has protein citrullination been implicated in rheumatoid arthritis? | Yes, protein citrullination been implicated in rheumatoid arthritis. | Protein citrullination is a posttranslational modification that has attracted
increased attention, especially for its involvement in rheumatoid arthritis
(RA). Here, we assess the citrullinome in RA synovial fluid by direct LC-MS/MS
analysis and by the use of an enrichment strategy based on citrulline specific
biotinylation. RA synovial fluid was depleted for abundant proteins, and total
and depleted fractions were analyzed. Frequency of citrullinated peptides and
their degree of citrullination could be determined for four known RA
autoantigens, as well as a novel in vivo autocitrullination site of
peptidylarginine deiminase 4. From the analysis of total and depleted synovial
fluid after enrichment we could estimate the numbers of citrullinated peptides
to be approximately 3600 and 2100, respectively. However, identification of
these biotinylated peptides by MS/MS turned out to be very difficult due to
fragmentation of the biotin moiety. By direct MS analysis of the total and
depleted synovial fluid without enrichment, 119 and 157 citrullinated peptides
were identified, respectively. This indicates that direct analysis allows
identification of only a fraction of the citrullinated proteins present in
synovial fluid and that specific enrichment is still needed for a comprehensive
in-depth elucidation of the citrullinome. Autoantibodies have been associated with human pathologies for a long time,
particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known
since the late 1930s to be associated with rheumatoid arthritis (RA). The
discovery of anticitrullinated protein antibodies in the last century has
changed this and other posttranslational modifications (PTM) relevant to RA have
since been described. Such PTM introduce neoepitopes in proteins that can
generate novel autoantibody specificities. The recent recognition of these novel
specificities in RA provides a unique opportunity to understand human B-cell
development in vivo. In this paper, we will review the three of the main classes
of PTMs already associated with RA: citrullination, carbamylation, and
oxidation. With the advancement of research methodologies it should be expected
that other autoantibodies against PTM proteins could be discovered in patients
with autoimmune diseases. Many of such autoantibodies may provide significant
biomarker potential. Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid
arthritis (RA). However, the direct effects of CII citrullination on cell
behavior have not been described. To study whether citrullination of CII could
affect cellular functions, we measured the adhesion of 3 different cell types
(human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal
stem cells) with impedance-based technology. The binding of different collagen
receptor integrins to citrullinated collagen was studied by CHO cell lines, each
overexpressing 1 of the 4 human collagen receptors on the cell surface, and with
solid-phase binding assays, using the recombit human integrin α1I, α2I, α10I,
and α11I domains. Collagen citrullination decreased the adhesion of synovial
fibroblasts ∼50% (P<0.05) and mesenchymal stem cells ∼40% (P<0.05) by
specifically decreasing the binding of integrins α10β1 and α11β1 to
arginine-containing motifs, such as GFOGER. In contrast, citrullination had only
a minor effect on the function of α1β1 and α2β1 integrins, which have been
reported to play a critical role in regulating leukocyte function. Molecular
modeling was used to explain the detected functional differences at the
structural level. Given that the integrins regulate cell metabolism,
proliferation, and migration, we suggest that collagen citrullination modifies
the pathogenesis of RA. Here, CII citrullination was shown to decrease the
survival of mesenchymal stem cells. Protein post-translational modifications like glycation, carbamylation and
citrullination increase the functional diversity of the proteome but in disease
situations might do more harm than good. Post-translational modifications of ECM
proteins are thus appearing as mechanisms, which contribute to tissue
dysfunction in chronic kidney disease, in diabetes and in various inflammatory
diseases. In chronic renal failure, carbamylation could lead to kidney fibrosis.
In diabetes, high glucose levels lead to non-enzymatic glycation and
cross-linking of collagens, which contribute to tissue stiffening with
consequences for cardiovascular and renal functions. In inflammatory diseases,
citrullination deiminates arginine residues with possible consequences for
integrin-mediated cell adhesion to RGD- and GFOGER sequences in ECM proteins.
Citrullination of fibronectin was in one study suggested to affect cell adhesion
by modifying the heparin-binding site and not the RGD site. In a recent
publication citrullination of GFOGER sequences in collagen II was demonstrated
to selectively affect α10β1 and α11β1 integrin-mediated cell adhesion to
collagen II, with consequences for synovial fibroblast and stem cell adhesion
and migration. The implications of citrullination affecting integrin binding in
disease open up a new area of study and might have implications for the
pathogenesis of inflammatory diseases like rheumatoid arthritis and
periodontitis. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the
presence of rheumatoid factor (RF) and anti-citrullinated protein/peptide
autoantibodies (ACPAs). Citrulline derives from arginine by peptidyl arginine
deiminases, and ACPAs are directed against different citrullinated antigens,
including fibrinogen, fibronectin, α-enolase, collagen type II, histones. ACPAs
are present in two thirds of RA patients have higher specificity than RF for RA,
and are associated with joint radiographic damage and extra-articular
manifestations and they are detected years before the onset clinical arthritis.
Recent studies suggest that citrullinated antigens are most likely arthritogenic
autoantigens in RA. ACPA production is associated with the HLA-DRB1 shared
epitope (HLA-DRB1 SE) and accounts for the well-known RA-HLA-DRB1 SE
association, as T cells recognize citrullinated peptides. Smoking and
periodontitis, known environmental risk factors for RA promote protein
citrullination and ACPA production. Cirullinated proteins are capable of
inducing arthritis in transgenic mice carrying HLA-DRB1 SE genes, and ACPAs
induce macrophage TNF-α production, osteoclastogenesis and complement
activation. They also induce the formation of neutrophil extracellular traps
(NETs). NETs, increased in RA, are a source of citrullinated autoantigens in RA
and induce fibroblast interleukin-8 production. This knowledge is likely to have
therapeutic implications, as there is a need of matching therapy with patient
profile. Abatacept, a T cell activation modulator, is the best therapy for
ACPA(+) RA patients, although clinical data are sparse at present. Rituximab, a
monoclonal antibody that depletes B cells, is also the best therapy for ACPA(+)
RA patients, and clinical data support this view. The conversion of an arginine residue in a protein to a citrulline residue, a
reaction carried out by enzymes called peptidylarginine deiminases (PADs), is
rather subtle. One of the terminal imide groups in arginine is replaced by
oxygen in citrulline, thus resulting in the loss of positive charge and the gain
of 1 dalton. This post-translational modification by PAD enzymes is conserved in
vertebrates and affects specific substrates during development and in various
mature cell lineages. Citrullination offers a unique perspective on autoimmunity
because PAD activity is stringently regulated, yet autoantibodies to
citrullinated proteins predictably arise. Autoantigens recognized by
anti-citrullinated protein antibodies (ACPA) include extracellular proteins such
as filaggrin, collagen II, fibrinogen, and calreticulin; membrane-associated
proteins such as myelin basic protein; cytoplasmic proteins such as vimentin and
enolase; and even nuclear proteins such as histones. Some ACPA are remarkably
effective as diagnostics in autoimmune disorders, most notably rheumatoid
arthritis (RA). Several ACPA can be observed before other clinical RA
manifestations are apparent. In patients with RA, ACPA may attain a sensitivity
that exceeds 70 % and specificity that approaches 96-98 %. The biological
context that may account for the induction of ACPA emerges from studies of the
cellular response of the innate immune system to acute or chronic stimuli. In
response to infections or inflammation, neutrophil granulocytes activate PAD,
citrullinate multiple autoantigens, and expel chromatin from the cell. The
externalized chromatin is called a neutrophil extracellular "trap" (NET).
Citrullination of core and linker histones occurs prior to the release of
chromatin from neutrophils, thus implicating the regulation of citrullinated
chromatin release in the development of autoreactivity. The citrullination of
extracellular autoantigens likely follows the release of NETs and associated
PADs. Autoantibodies to citrullinated histones arise in RA, systemic lupus
erythematosus, and Felty's syndrome patients. The citrullination of linker
histone H1 may play a key role in NET release because the H1 histone regulates
the entry and exit of DNA from the nucleosome. Juxtaposition of citrullinated
histones with infectious pathogens and complement and immune complexes may
compromise tolerance of nuclear autoantigens and promote autoimmunity. INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse
the posttranslational conversion of peptidylarginine to peptidylcitrulline.
Citrullination of proteins is well described in rheumatoid arthritis (RA), and
hypercitrullination of proteins may be related to inflammation in general. PAD
activity has been demonstrated in various cell lysates, but so far not in
synovial fluid. We aimed to develop an assay for detection of PAD activity, if
any, in synovial fluid from RA patients.
METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the
immobilized substrate for citrullination and anti-citrullinated fibrinogen
antibody as the detecting agent were used for measurement of PAD activity in
synovial fluid samples from five RA patients. The concentrations of PAD2 and
calcium were also determined.
RESULTS: Approximately 150 times lower levels of recombit human PAD2 (rhPAD2)
than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was
detected in four of five synovial fluid samples from RA patients and correlated
with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium
requirement for half-maximal activities of PAD2 and PAD4 were found in a range
from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium
levels for the citrullination process to occur.
CONCLUSIONS: We present an assay with high specificity for PAD2 activity and
show that citrullination of fibrinogen can occur in cell-free synovial fluid
from RA patients. Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint
inflammation and extra-articular manifestations. To prevent progressive and
irreversible structural damage, early diagnosis of RA is of paramount
importance. Antibodies directed against citrullinated proteins and peptides
(ACPAs) are the most specific serological markers available for diagnosing RA.
ACPAs may be detected several years before symptoms appear, and their presence
at disease onset is a good predictor of the development of erosive joint
lesions. Synthetic peptides can replace cognate proteins in solid-phase assays
for specific autoantibody recognition in RA patients. The use of synthetic
peptides instead of proteins represents an advantage in terms of the
reproducibility of such immunoassays. They give absolute control over the exact
epitopes presented. Furthermore, it is difficult to prepare sufficient amounts
of high-quality antigenic proteins with a well-defined degree of citrullination.
Synthetic citrullinated peptides, in contrast, are easily obtained in a pure
form with a well-defined chemical structure and the epitopes can be precisely
oriented in the plate by covalent binding of the peptides. We have recently
obtained and highlighted the application of chimeric peptides bearing different
citrullinated protein domains for the design of RA diagnosis systems. Our
results indicate that more than one serological test is required to classify RA
patients based on the presence or absence of ACPAs. Each of the target molecules
reported (fibrin, vimentin and filaggrin) helps to identify a particular subset
of RA patients. |
Show results of randomised controlled trials for certolizumab pegol. | Improvement of clinical results (ACR50, 28 joint disease activity score (DAS-28) remission and HAQ scores) with certolizumab pegol. Adverse events were more frequent with certolizumab; there was a statistically significant increase in the number of serious adverse events, infections and hypertension.
Randomised controlled trials (RCTs) of CZP have demonstrated rapid improvements in workplace and home productivity. | This paper presents a summary of the evidence review group (ERG) report into the
clinical effectiveness and cost-effectiveness of certolizumab pegol (CZP) for
adults with active rheumatoid arthritis (RA) that have not responded adequately
to treatment with conventional disease modifying anti-rheumatic drugs (DMARDs)
including methotrexate (MTX), in accordance with the licensed indication, based
upon the evidence submission from the manufacturer to the National Institute for
Health and Clinical Excellence (NICE) as part of the single technology appraisal
(STA) process. The outcome measures included American College of Rheumatology
(ACR) 20, 50 and 70 response rates and quality of life measures after 3 months
and 6 months of treatment. The ERG examined the submission's search strategies
and considered they appeared comprehensive and that it was unlikely that
relevant studies would have been missed. Only English language studies were
considered in the submission and non-English language studies relevant to the
decision problem may possibly have been ignored. The ERG analysed the first
submitted economic model so as to itemise in detail clarification points that
were brought to the attention of the manufacturer. In response the manufacturer
submitted a modified cost-effectiveness analysis. The ERG undertook further
analysis of this second model and other additional submitted evidence. The
clinical evidence was derived from two multicentre blinded randomised controlled
trials (RCTs) comparing CZP + MTX to placebo + MTX (the RAPID 1 and RAPID 2
trials). RAPID 1 lasted 52 weeks with 982 patients and RAPID 2 24 weeks with 619
patients. Evidence for clinical effectiveness of CZP in mono-therapy came from
the 24-week FAST4WARD trial with 220 patients that compared CZP (400 mg every 4
weeks) versus placebo. The three key RCTs demonstrated statistically significant
superiority of CZP + MTX versus placebo + MTX and of CZP versus placebo with
respect to a variety of outcomes including ACR 20, ACR 50 and ACR 70 measures
and quality of life measures at 3 and 6 months. On the basis of results from the
indirect comparison meta-analyses, the manufacturer suggested that CZP may be at
least as effective as other 'biological' DMARD (bDMARD) comparators and, in a
few ACR measures at 3 and 6 months, more effective. CZP is an effective therapy
for adult RA patients whose disease has failed to respond adequately to cDMARDs
including MTX or who are intolerant of MTX. The cost-effectiveness of CZP
relative to other bDMARDs is unclear because the economic modelling undertaken
may have ignored relevant effectiveness data and potential differences between
trial populations, and so may have included effectiveness results that were
biased in favour of CZP; underestimated uncertainty in the relative
effectiveness of compared DMARDs; and ignored the potential influence of
differences between bDMARDs with regard to adverse events and their related
costs and health impacts. The NICE guidance issued in October 2009 states that:
the Committee is minded not to recommend certolizumab pegol as a treatment
option for people with RA; and the Committee recommends that NICE asks the
manufacturer of CZP for more information on the clinical effectiveness and
cost-effectiveness of CZP for the treatment of people with RA. On receipt of
this information and details of a patient access scheme NICE issued final
guidance recommending CZP, under certain criteria, as a treatment option for
people with RA. INTRODUCTION: Improved understanding of the pathogenesis of rheumatoid arthritis
(RA), and subsequent development of targeted therapies, have greatly advanced
the management of this chronic inflammatory disease. The aim of treatment is a
state of clinical remission. Certolizumab pegol (CZP) is a novel pegylated TNF
alpha inhibitor (TNFi) therapy and is the focus of this review.
AREAS COVERED: CZP is different from other TNFi as it contains a polyethylene
glycol (PEG) moiety, and lacks the constant fragment (Fc) of immunoglobulin;
therefore it does not activate complement. In this review in addition to
clinical efficacy of CZP, effects on radiographic and patient-reported outcomes,
are discussed. Adverse event data from clinical trials and extension studies are
also reviewed.
EXPERT OPINION: The addition of novel TNFi therapies to treat RA is welcomed. As
well as displaying clinical efficacy, there is evidence to suggest that CZP has
unique characteristics, including reduced transfer across the placenta and
reduced frequency of injection site reactions. Furthermore, randomised
controlled trials (RCTs) of CZP have demonstrated rapid improvements in
workplace and home productivity in patients contributing to reducing the
significant socioeconomic burden of RA. |
Which is the mass-tag that reveal the ubiquitination of a lysine residue? | Lys-ɛ-Gly-Gly (K-ɛ-GG) is the remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains. | The quantitation of lysine post-translational modifications (PTMs) by bottom-up
mass spectrometry is convoluted by the need for analogous derivatives and the
production of different tryptic peptides from the unmodified and modified
versions of a protein. Chemical derivatization of lysines prior to enzymatic
digestion circumvents these problems and has proven to be a successful method
for lysine PTM quantitation. The most notable example is the use of
deuteroacetylation to quantitate lysine acetylation. In this work, levels of
lysine ubiquitination were quantitated using a structurally homologous label
that is chemically similar to the diglycine (GlyGly) tag, which is left at the
ubiquitination site upon trypsinolysis. The LC-MS analysis of a chemically
equivalent monoglycine (Gly) tag that is analogous to the corresponding GlyGly
tag proved that the monoglycine tag can be used for the quantitation of
ubiquitination. A glycinylation protocol was then established for the
derivatization of proteins to label unmodified lysine residues with a single
glycine tag. Ubiquitin multimers were used to show that after glycinylation and
tryptic digestion, the mass spectrometric response from the corresponding
analogous tagged peptides could be compared for relative quantitation. For a
proof of principle regarding the applicability of this technique to the analysis
of ubiquitination in biological samples, the glycinylation technique was used to
quantitate the increase in monoubiquitinated histone H2B that is observed in
yeast which lacks the enzyme responsible for deubiquitinating H2B-K123, compared
to wild-type yeast. Ubiquitination is essential for the regulation of cellular protein homeostasis.
It also has a central role in numerous signaling events. Recent advances in the
production and availability of antibodies that recognize the Lys-ɛ-Gly-Gly
(K-ɛ-GG) remt produced by trypsin digestion of proteins having ubiquitinated
lysine side chains have markedly improved the ability to enrich and detect
endogenous ubiquitination sites by mass spectrometry (MS). The following
protocol describes the steps required to complete a large-scale ubiquitin
experiment for the detection of tens of thousands of distinct ubiquitination
sites from cell lines or tissue samples. Specifically, we present detailed,
step-by-step instructions for sample preparation, off-line fractionation by
reversed-phase chromatography at pH 10, immobilization of an antibody specific
to K-ɛ-GG to beads by chemical cross-linking, enrichment of ubiquitinated
peptides using these antibodies and proteomic analysis of enriched samples by
LC-tandem MS (MS/MS). Relative quantification can be achieved by performing
stable isotope labeling by amino acids in cell culture (SILAC) labeling of
cells. After cell or tissue samples have been prepared for lysis, the described
protocol can be completed in ∼5 d. Ubiquitination is a process that involves the covalent attachment of the
76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine
(K) residues on substrate proteins. This post-translational modification elicits
a wide range of functional consequences including targeting proteins for
proteasomal degradation, altering subcellular trafficking events, and
facilitating protein-protein interactions. A number of methods exist for
identifying the sites of ubiquitination on proteins of interest, including
site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS).
Recent publications have also highlighted the use of peptide-level
immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for
global characterization of ubiquitination sites. Here we investigated the
utility of this technique for focused mapping of ubiquitination sites on
individual proteins. For a series of membrane-associated and cytoplasmic
substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α
(TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently
yielded additional ubiquitination sites beyond those identified in protein level
AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were
prepared and used to compare the abundances of individual K-GG peptides from
samples prepared in parallel. Consistently, K-GG peptide immunoaffinity
enrichment yielded greater than fourfold higher levels of modified peptides than
AP-MS approaches. Using this approach, we went on to characterize inducible
ubiquitination on multiple members of the T-cell receptor complex that are
functionally affected by endoplasmic reticulum (ER) stress. Together, these data
demonstrate the utility of immunoaffinity peptide enrichment for single protein
ubiquitination site analysis and provide insights into the ubiquitination of
HER2, DVL2, and proteins in the T-cell receptor complex. The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and
breast cancer cells. Its activity was mapped to the 162 amino acid propeptide
domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the
Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven
breast tumor burden in a xenograft model. Since its mechanism of action is
largely unknown, co-affinity-purification/mass spectrometry was performed and
the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating
protein. CIN85 is an SH3-containing adapter protein that is overexpressed in
invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3
ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest
affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to
the AMAP1 complex where its ubiquitination activity is necessary for cancer
cells to develop an invasive phenotype and to degrade the matrix. Direct
interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation
analysis of lysates from breast cancer cells and of purified expressed proteins.
CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85
regions and deletion mutants of LOX-PP were prepared and used to map the sites
of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino
acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and
R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl.
Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this
region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the
invasive phenotype of breast cancer cells, including their ability to degrade
the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP
interacts with CIN85 via a novel SH3-binding motif and this association reduces
CIN85-promoted invasion by breast cancer cells. The type II iodothyronine deiodinase (D2) is a type I endoplasmic reticulum
(ER)-resident thioredoxin fold-containing selenoprotein that activates thyroid
hormone. D2 is inactivated by ER-associated ubiquitination and can be
reactivated by two ubiquitin-specific peptidase-class D2-interacting
deubiquitinases (DUBs). Here, we used D2-expressing cell models to define that
D2 ubiquitination (UbD2) occurs via K48-linked ubiquitin chains and that
exposure to its natural substrate, T4, accelerates UbD2 formation and
retrotranslocation to the cytoplasm via interaction with the p97-ATPase complex.
D2 retrotranslocation also includes deubiquitination by the p97-associated DUB
Ataxin-3 (Atx3). Inhibiting Atx3 with eeyarestatin-I did not affect D2:p97
binding but decreased UbD2 retrotranslocation and caused ER accumulation of
high-molecular weight UbD2 bands possibly by interfering with the
D2-ubiquitin-specific peptidases binding. Once in the cytosol, D2 is delivered
to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit
S5a and increased colocalization with the 20S proteasome. We conclude that
interaction between UbD2 and p97/Atx3 mediates retranslocation of UbD2 to the
cytoplasm for terminal degradation in the proteasomes, a pathway that is
accelerated by exposure to T4. Protein ubiquitination occurs through formation of an isopeptide bond between
the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate
lysine residue. This post-translational modification, which occurs through the
attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin
chains, is involved in crucial cellular events such as protein degradation,
cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin
pathways is also implicated in the pathogenesis of several human diseases
ranging from cancer to neurodegeneration. However, despite the undoubted
biological importance, understanding the molecular basis of how ubiquitination
regulates different pathways has up to now been strongly limited by the
difficulty of producing the amounts of highly homogeneous samples that are
needed for a structural characterization by X-ray crystallography and/or NMR.
Here, we report on the production of milligrams of highly pure Josephin
mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic
ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein
responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first
deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination.
We demonstrate that the samples produced with the described method are correctly
folded and suitable for structural studies. The protocol allows facile selective
labelling of the components. Our results provide an important proof-of-concept
that may pave the way to new approaches to the in vitro study of ubiquitinated
proteins. |
Which gene is involved in CADASIL? | Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of familial vascular dementia, is caused by mutations of the NOTCH3 gene. | Cerebral autosomal domit arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL) is a vascular dementing disease caused by
mutations in the NOTCH3 gene, most which are missense mutations leading to an
uneven number of cysteine residues in epidermal growth factor-like repeats in
the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by
degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on
the VSMCs of small and middle-sized arteries. Recent studies have demonstrated
that impairment of Notch3 signaling is not the primary cause of the disease. In
the present study we used proteomic analysis to characterize the protein
expression pattern of a unique material of genetically genuine cultured human
CADASIL VSMCs. We identified 11 differentially expressed proteins, which are
involved in protein degradation and folding, contraction of VSMCs, and cellular
stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic
reticulum stress and activation of unfolded protein response, leading to
increased reactive oxygen species and inhibition of cell proliferation. In
addition, upregulation of contractile proteins suggests an alteration in the
signaling system of VSMC contraction. The accumulation of N3ECD on the cell
surface possibly upregulates the angiotensin II regulatory feedback loop and
thereby enhances the readiness of the cells to respond to angiotensin II
stimulation. Le CADASIL, ou Cerebral autosomal domit arteriopathy with subcortical
infarcts and Ieukoencephalopathy est une affection héréditaire des petites
artères cérébrabes survet chez l'adulte d'âge moyen, due à des mutations du
gène Notch3. La maladie est responsable de lésions diffuses de la substance
blanche associées à des infarctus lacunaires au niveau des régions
sous-corticales cérébrales. Elle est à l'origine de crises de migraine avec
aura, d'accidents ischémiques cérébraux et est associée à différents degrés
d'altération cognitive et à des troubles de l'humeur. CADASIL est considéré
comme un modébe unique d'étude des «démences sous-corticales d'origine
ischémique». Des données récentes suggèrent que le nombre d'infarctus lacunaires
et la sévérité de l'atrophie cérébrale sont les principaux marqueurs de la
maladie associés au handicap cognitif et moteur de la maladie. Les troubles de
l'humeur sont rapportés par 10 à 20% des patients, le plus souvent en
association avec des altérations cognitives. Leur origine exacte demeure
indéterminée, la présence de lésions ischémiques au niveau des noyaux gris ou au
sein de la substance blanche frontale pourrait favoriser l'apparition de ces
symptômes. Des études complémentaires sont nécessaires pour mieux comprendre les
relations entre les lésions cérébrales et les symptômes cognitifs et
psychiatriques observés au cours de cette maladie des petits vaisseaux du
cerveau. Proteins of the Notch family are cell surface receptors that transduce signals
between neighbouring cells. The Notch signalling pathway is highly
evolutionarily conserved and critical for cell fate determination during
embryonic development, including many aspects of vascular development. The
interaction of Notch receptors with ligands leads to cleavage of the Notch
intracellular domain (NICD) which then translocates to the nucleus and activates
the transcription factor CBF1/JBP-Jkappa, regulating downstream gene expression.
To date four Notch receptors have been found in mammals. Of these, Notch3 is
predomitly expressed in adult arterial smooth muscle cells in human. NOTCH3
gene mutations cause the autosomal domit condition, cerebral autosomal
domit arteriopathy with subcortical infarcts and leukoecephelopathy
(CADASIL), an inherited early stroke syndrome leading to dementia due to
systemic vascular degeneration. This suggests that Notch3 plays a critical role
in maintaining the phenotypic stability of vascular smooth muscle cells (VSMCs).
Recent publications indicate that Notch3 is involved in vascular injury and is a
determit of VSMC survival, but its exact function is unknown. The molecular
mechanisms underlying CADASIL pathology are therefore intriguing. Investigation
of CADASIL mutant Notch3 shows that the majority of mutations do not change
CBF1/JBP-Jkappa mediated classic Notch activation, so the pathological
consequences of NOTCH3 mutations in CADASIL patients can not be simply explained
by loss- or gain-of-function in the classic Notch signalling pathway. This
suggests that a novel Notch3-mediated signalling pathway may be present in
VSMCs, or cross-regulation of Notch3 to other signalling pathway(s) may play a
critical role on VSMCs survival. Alternatively, the mutant Notch3 may gain a
novel or toxic function in VSMCs. This review will focus on recent findings of
Notch3 in vascular development and in regulating the VSMC behaviour and
phenotype, and will use findings on investigating the molecular pathology of the
single gene disorder CADASIL to understand the function of Notch3 in VSMCs. BACKGROUND: Cerebral autosomal domit arteriopathy with subcortical infarcts
and leukoencephalopathy (CADASIL) is increasingly recognized as an inherited
arterial disease leading to a step-wise decline and eventually to dementia.
CADASIL is caused by mutations in NOTCH3 epidermal growth factor-like repeat
that maps to chromosome 19. CADASIL cases have been identified in most countries
of Western and Central Europe, the Americas, Japan, Australia, the Caribbean,
South America, Tanzania, Turkey, South Africa and Southeast Asia, but not in
Arabs.
METHODS: We studied three families from Saudi Arabia (Family A), Kuwait (Family
B) and Yemen (Family C) with 19 individuals affected by CADASIL.
RESULTS: The mean age of onset was 31 +/- 6 and the clinical presentation
included stroke in 68%, subcortical dementia in 17% and asymptomatic
leukoariosis detected by MRI in 15%. Migraine and depression were frequently
associated, 38% and 68% respectively. The mean age of death was 56 +/- 11. All
NOTCH3 exons were screened for mutations, which revealed the presence of
previously reported mutations c.406C>T (p.Arg110>Cys) in two families (family
A&B) and c.475C>T (p.Arg133>Cys) mutation in family C.
CONCLUSION: CADASIL occurs in Arabs, with clinical phenotype and genotype
similar to that in other ethnic groups. To investigate the migraine locus around the C19p13 region through analysis of
the NOTCH3 gene (C19p13.2-p13.1), previously shown to be a gene involved in
CADASIL and the TNFSF7 gene (C19p13), homologous to the ligands of TNF-alpha and
TNF-beta, genes that have previously been associated with migraine. The NOTCH3
gene was analysed by sequencing all exons with known CADASIL mutations in a
typical (non-familial hemiplegic) migraine family (MF1) that has previously been
shown to be linked to C19p13. The TNFSF7 gene was investigated through SNP
association analysis using a matched case-control migraine population. NOTCH3
gene sequencing results for affected members of MF1 proved to be negative for
all known sequence variants giving rise to mutations for CADASIL. TNFSF7 gene
chi-square results showed non-significant P values across all populations tested
against controls, except for the MO subgroup which displayed a possible
association with the TNFSF7 SNP (genotype, allele analysis P = 0.036, P = 0.017
respectively). Our results suggest that common migraine is not caused by any
known CADASIL mutations in the NOTCH3 gene of interest. However, the TNFSF7 gene
displayed signs of involvement in a MO affected population and indicates that
further independent studies of this marker are warranted. Cerebral autosomal domit arteriopathy with subcortical infarcts and
leucoencephalopathy (CADASIL) is the most common heritable cause of stroke and
vascular dementia in adults. Clinical and neuroimaging features resemble those
of sporadic small-artery disease, although patients with CADASIL have an earlier
age at onset of stroke events, an increased frequency of migraine with aura, and
a slightly variable pattern of ischaemic white-matter lesions on brain MRI.
NOTCH3 (Notch homolog 3), the gene involved in CADASIL, encodes a transmembrane
receptor primarily expressed in systemic arterial smooth-muscle cells.
Pathogenetic mutations alter the number of cysteine residues in the
extracellular domain of NOTCH3, which accumulates in small arteries of affected
individuals. Functional and imaging studies in cultured cells, genetically
engineered mice, and patients with CADASIL have all provided insights into the
molecular and vascular mechanisms underlying this disease. A recent multicentre
trial in patients with cognitive impairment emphasises the feasibility of
randomised trials in patients with CADASIL. In this Review, we summarise the
current understanding of CADASIL, a devastating disorder that also serves as a
model for the more common forms of subcortical ischaemic strokes and pure
vascular dementia. We reviewed the characteristics of headache in patients with cerebral autosomal
domit arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL), to verify the appropriateness of the International Classification of
Headache Disorders, second edition (ICHD-II) criteria. Available data were found
through Medline/PubMed using the keyword "cerebral autosomal domit
arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)". The
search was restricted to studies published in English in the years between 1993
and 2008. We excluded studies that did not report original data on CADASIL and
information regarding the presence of headache. We found 34 studies reporting
data on 749 patients overall; 387 (51.7%) patients had headache. According to
the authors' definition, 356 (92%) patients were reported as having migraine and
31 (8%) as having headache. Of the 356 patients who were defined as migraineurs,
125 (35.1%) had migraine with aura, 7 (2%) migraine without aura, 156 (43.8%)
unspecified migraine and 68 (19.1%) had more than one type of migraine. Among
the 31 patients reported as suffering from headache, the headache was not
further detailed in 18 (58.1%) patients; it was defined as chronic in 6 (19.3%),
as resembling migraine with aura in 4 (12.9%), as resembling migraine without
aura in 2 (6.5%) and as tension type in 1 (3.2%) patient. In patients with
CADASIL, the headache was usually referred to as migraine and mostly as migraine
with aura. However, this referral is formally incorrect since the diagnostic
criteria for any type of migraine in the ICHD-II require that the disturbance is
not attributed to another disorder. For this reason, we suggest updating the
ICHD-II in relation to CADASIL. Our suggestion is to insert a new category
referred to as Headache attributed to genetic disorder including Headache
attributed to CADASIL. Cerebral autosomal domit arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene and is
clinically characterized by recurrent stroke, cognitive decline, psychiatric
disturbances and migraine. The prevalence of migraine in CADASIL is slightly
higher than in the general population, and the proportion of migraine with aura
is much higher. The pathophysiological mechanism that leads to increased aura
prevalence in CADASIL is unknown. Possible mechanisms of the excess of migraine
with aura are an increased susceptibility to cortical spreading depression (CSD)
or a different expression of CSD. It is also possible that the brainstem
migraine area is involved in CADASIL. Last, it is possible that the NOTCH3
mutation acts as a migraine aura susceptibility gene by itself. In this
narrative review we summarize the literature about migraine in CADASIL, with a
special focus on what CADASIL might teach us about the pathophysiology of
migraine. Notch3 is a single pass transmembrane protein belonging to the Notch receptor
family. Notch proteins are involved in a very conserved signaling system (Notch
signaling) with a broad spectrum of functions, from cell proliferation and
differentiation to apoptosis. Mutations in Notch3 gene are linked to cerebral
autosomal domit arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL), a disorder characterized by stroke and dementia
in young adults. Studies evaluating Notch3 expression in human differentiated
cells and adult tissues have shown high Notch3 levels only in vascular smooth
muscle cells (VSMC). However, it has been hypothesized that Notch3 is
ubiquitously expressed in adult human tissues. Our aim was to evaluate Notch3
expression in human peripheral blood lymphocytes (PBLs) and fibroblasts from
normal healthy subjects. In both cell types, we examined the expression of
Notch3 by reverse transcriptase-polymerase chain reaction (RT-PCR) and
quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, we
assessed Notch3 protein expression by Western blot analysis. RT-PCR and qRT-PCR
analysis showed the presence of Notch3 mRNA in both cell types. Western blot
analysis confirmed Notch3 protein expression in PBLs and fibroblasts though
showing different profiles. Our data support the expression of Notch3 in adult
human cell types, and suggests that PBLs and fibroblasts could provide readily
available cells for the study of the role of Notch3 expression in the
pathogenetic mechanisms leading to different human disease. Cerebrolysin (Cere) is a peptidergic nootropic drug with neurotrophic properties
which has been used to treat dementia and sequelae of stroke. Use of Cere
prevents nuclear structural changes typical of apoptosis and significantly
reduces the number of apoptotic cells after several apoptotic stimuli. Cerebral
Autosomal Domit Arteriopathy with Subcortical Infarcts and
Leukoencephalopathy (CADASIL) is a hereditary disease caused by mutations of the
Notch3 gene encoding the Notch3 protein. Notch3 is involved in the regulation of
apoptosis, modulating Fas-Ligand (Fas-L)- induced apoptosis. The aim of this
study was to evaluate the in vitro protective effects of Cere against oxidative
stress-induced apoptosis in cells from CADASIL patients. We used peripheral
blood lymphocytes (PBLs) from 15 CADASIL patients (age range 34-70 years);
2-deoxy-D-ribose (dRib), a highly reducing sugar, was used as paradigm
pro-apoptotic stimulus. Apoptosis was analyzed by flow cytometry and
fluorescence microscopy. Administration of Cere to PBLs from CADASIL patients
cultured under standard conditions had no effect on the percentage of apoptotic
cells. Administration of Cere to PBLs cultured with dRib caused a significant
decrease in apoptosis after 48 h of culture in only 5 patients, whereas in the
other 10 patients, Cere treatment was not associated with any significant
difference in the percentage of apoptosis. This result showed a protective
effect of Cere against oxidative stress-induced apoptosis only in 30 % of the
CADASIL patients, suggesting that the Notch3 gene probably does not influence
the anti-apoptotic properties of Cere in vitro. |
What is the role of thyroid hormone receptor alpha1 in insulin secretion? | Liganded TR(alpha) plays a critical role in beta-cell replication and in expansion of the beta-cell mass. the TRalpha P398H mutation which cannot bind T3, is associated with insulin resistance. Loss of Thra protects mice from high-fat diet-induced hepatic and peripheral insulin resistance. | Thyroid hormone has profound effects on metabolic homeostasis, regulating both
lipogenesis and lipolysis, primarily by modulating adrenergic activity. We
generated mice with a point mutation in the thyroid hormone receptor alpha
(TRalpha) gene producing a domit-negative TRalpha mutant receptor with a
proline to histidine substitution (P398H). The heterozygous P398H mutant mice
had a 3.4-fold (p < 0.02) increase in serum thyrotropin (TSH) levels. Serum
triiodothyronine (T3) and thyroxine (T4) concentrations were slightly elevated
compared with wild-type mice. The P398H mice had a 4.4-fold increase in body fat
(as a fraction of total body weight) (p < 0.001) and a 5-fold increase in serum
leptin levels (p < 0.005) compared with wild-type mice. A 3-fold increase in
serum fasting insulin levels (p < 0.002) and a 55% increase in fasting glucose
levels (p < 0.01) were observed in P398H compared with wild-type mice. There was
a marked reduction in norepinephrine-induced lipolysis, as reflected in reduced
glycerol release from white adipose tissue isolated from P398H mice. Heart rate
and cold-induced adaptive thermogenesis, mediated by thyroid
hormone-catecholamine interaction, were also reduced in P398H mice. In
conclusion, the TRalpha P398H mutation is associated with visceral adiposity and
insulin resistance primarily due to a marked reduction in
catecholamine-stimulated lipolysis. The observed phenotype in the TRalpha P398H
mouse is likely due to interference with TRalpha action as well as influence on
other metabolic signaling pathways. The physiologic significance of these
findings will ultimately depend on understanding the full range of actions of
this mutation. Failure of the functional pancreatic beta-cell mass to expand in response to
increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing
studies indicate that replication of existing beta-cells is important for
beta-cell proliferation in adult animals. In rat pancreatic beta-cell lines
(RIN5F), treatment with 100 nM thyroid hormone (triiodothyronine, T(3)) enhances
cell proliferation. This result suggests that T(3) is required for beta-cell
proliferation or replication. To identify the role of thyroid hormone receptor
alpha (TR(alpha)) in the processes of beta-cell growth and cell cycle
regulation, we constructed a recombit adenovirus vector, AdTR(alpha).
Infection with AdTR(alpha) to RIN5F cells increased the expression of cyclin D1
mRNA and protein. Overexpression of the cyclin D1 protein in
AdTR(alpha)-infected cells led to activation of the cyclin D1/cyclin-dependent
kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and
cell proliferation following treatment with 100 nM T(3). Conversely, lowering
cellular cyclin D1 by small interfering RNA knockdown in AdTR(alpha)-infected
cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited
cell proliferation. Furthermore, in immunodeficient mice with
streptozotocin-induced diabetes, intrapancreatic injection of AdTR(alpha) led to
the restoration of islet function and to an increase in the beta-cell mass.
These results support the hypothesis that liganded TR(alpha) plays a critical
role in beta-cell replication and in expansion of the beta-cell mass during
postnatal development. Thus, liganded TR(alpha) may be a target for therapeutic
strategies that can induce the expansion and regeneration of beta-cells. |
Do R-loops tend to form at sites of DNA replication? | R-loops co-localize with the ORC within the same CpG island region in a significant fraction of these efficient replication origins. Physiological R-loop formation at CpG island promoters can contribute to DNA replication origin specification at these regions, the most efficient replication initiation sites in mammalian cells. One mechanism may be that downstream of a replication block, RNA at R-loops is extended by DNA polymerase I, opening up the DNA duplex and leading to the recruitment of the replisome. This would allow replication to proceed while the original block is repaired or bypassed. Thus, the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery. | Escherichia coli rnhA mutants devoid of RNase HI exhibit constitutive stable DNA
replication, cSDR, which is thought to be initiated from R-loops stabilized in
the absence of RNase HI. We found that a combination of an rnhA and a recG
mutation is lethal to the cell. recG mutations that inactivate the helicase
activity of RecG protein and inhibit reverse branch migration of Holliday
junctions impart phenotypes resembling those of rnhA mutants. Thus, recG mutants
display cSDR activity, and recG polA double mutants are inviable as are rnhA
polA double mutants. These results suggest that the RecG helicase has a role in
preventing R-loop formation. A model that R-loops are formed by assimilation of
RNA transcripts into the duplex DNA is discussed. The model further postulates
that RecA protein catalyzes this assimilation reaction and that RecG protein
counteracts RecA in this reaction, resolves R-loops by its helicase activity, or
does both. Primers for vertebrate mitochondrial leading-strand DNA replication are products
of transcription synthesized by mitochondrial RNA polymerase. The precursor
primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed
during transcription through the replication origin (Xu, B., and Clayton, D. A.
(1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of
this primer RNA intermediate, we have used two methods to reconstitute model
R-loops containing the mouse mitochondrial DNA origin sequence. First, we
demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the
formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop
can be assembled by annealing presynthesized RNA and supercoiled DNA template in
the presence of formamide. R-loop formation by either method is dependent on
specific template sequences. The reconstituted R-loop is exceptionally stable
and exhibits an unexpected structure. Structural studies indicate that the RNA
strand is organized within the RNA-DNA base-paired region, suggesting that the
heteroduplex interaction occurs through a specific conformation. We propose that
the organized structure of the R-loop is critical for primer RNA function in
vivo with important implications for the RNA processing and DNA replication
machinery. The precursor primer RNA for mammalian mitochondrial DNA leading-strand
replication remains as a persistent R loop formed during transcription through
the mitochondrial DNA control region. We have examined model R loops, which
exist in a novel and physiologically accurate preprimer conformation, as
potential substrates for mammalian RNase mitochondrial RNA processing (MRP).
Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial
DNA origin. The multiple cleavage sites on the R-loop substrate match the
priming sites observed in vivo, suggesting that RNase MRP alone is capable of
generating virtually all of the leading-strand replication primers. Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the
RNA transcript can thread back onto the template DNA strand, resulting in an R
loop. Previously, we showed that initiation of R-loop formation at an R-loop
initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro
system with T7 RNA polymerase, we show that increased distance between the
promoter and the R-loop-supporting G-rich region reduces R-loop formation. When
the G-rich portion of the RNA transcript is downstream from the 5' end of the
transcript, the ability of this portion of the transcript to anneal to the
template DNA strand is reduced. When we nucleolytically resect the beginning of
the transcript, R-loop formation increases because the G-rich portion of the RNA
is now closer to the 5' end of the transcript. Short G-clustered regions can act
as RIZs and reduce the distance-induced suppression of R-loop formation.
Supercoiled DNA is known to favor transient separation of the two DNA strands,
and we find that this favors R-loop formation even in non-G-rich regions. Most
strikingly, a nick can serve as a strong RIZ, even in regions with no G
richness. This has important implications for class switch recombination and
somatic hypermutation and possibly for other biological processes in transcribed
regions. We have recently reported that topoisomerase 1 (Top1) cooperates with ASF/SF2, a
splicing factor of the SR family, to prevent unscheduled replication fork arrest
and genomic instability in human cells. Our results suggest that Top1 execute
this function by suppressing the formation of DNA-RNA hybrids during
transcription, these so-called R-loops interfering with the progression of
replication forks. Using ChIP-chip, we have shown that γ-H2AX, a marker of DNA
damage, accumulates at gene-rich regions of the genome in Top1-deficient cells.
This is best illustrated at histone genes, which are highly expressed during S
phase and display discrete γ-H2AX peaks on ChIP-chip profiles. Here, we show
that these γ-H2AX domains are different from those induced by camptothecin, a
Top1 inhibitor inducing double-strand DNA breaks throughout the genome. These
data support the view that R-loops promote genomic instability at specific sites
by blocking fork progression and inducing chromosome breaks. Whether this type
of transcription-dependent fork arrest contributes to the replication stress
observed in precancerous lesions is an important question that deserves further
attention. Bacteriophage T4 initiates DNA replication from specialized structures that form
in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on
(at least some) replication origins, with the annealed RNA serving as a primer
for leading-strand synthesis in one direction. As the infection progresses,
replication initiation becomes dependent on recombination proteins in a process
called recombination-dependent replication (RDR). RDR occurs when the
replication machinery is assembled onto D-loop recombination intermediates, and
in this case, the invading 3' DNA end is used as a primer for leading strand
synthesis. Over the last 15 years, these two modes of T4 DNA replication
initiation have been studied in vivo using a variety of approaches, including
replication of plasmids with segments of the T4 genome, analysis of replication
intermediates by two-dimensional gel electrophoresis, and genomic approaches
that measure DNA copy number as the infection progresses. In addition,
biochemical approaches have reconstituted replication from origin R-loop
structures and have clarified some detailed roles of both replication and
recombination proteins in the process of RDR and related pathways. We will also
discuss the parallels between T4 DNA replication modes and similar events in
cellular and eukaryotic organelle DNA replication, and close with some current
questions of interest concerning the mechanisms of replication, recombination
and repair in phage T4. During S phase, the replisome has to overcome many physical obstacles that can
cause replication fork stalling and compromise genome integrity. Transcription
is an important source of replicative stress and consequently, maintece of
genome integrity requires the protection of chromosomes from the deleterious
effects arising from the interaction between nascent RNAs and template DNA,
leading to stable DNA-RNA hybrids (R-loop) formation. We previously reported the
essential role of Omcg1 (Ovum Mutant Candidate Gene) for cell cycle progression
during early embryonic development. Here, we show that OMCG1 is a target of the
cell cycle checkpoint kinases ATR/ATM and is essential for S phase progression
in mouse embryonic fibroblasts. Using a conditional gene inactivation strategy,
we demonstrate that OMCG1 depletion impairs cell viability as a consequence of
DSB formation, checkpoint activation and replication fork collapse. We also show
that no chromosome breaks were generated in non-cycling Omcg1-deficient cells.
Furthermore, increased RNaseH expression significantly alleviated genomic
instability in deficient fibroblasts suggesting that cotranscriptional R-loops
formation contributes to the genesis of replication-dependent DSBs in these
cells. Together with recent reports describing its participation to complexes
involved in cotanscriptional processes, our results suggest that OMCG1 plays a
role in the tight coupling between mRNA processing pathways and maintece of
genome integrity during cell cycle progression. Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in
organisms from bacteria to humans. These structures have been shown in
eukaryotes to result in DNA damage and rearrangements; however, the mechanisms
underlying these effects have remained largely unknown. To investigate this, we
first show that R-loop formation induces chromosomal DNA rearrangements and
recombination in Escherichia coli, just as it does in eukaryotes. More
importantly, we then show that R-loop formation causes DNA replication fork
stalling, and that this in fact underlies the effects of R loops on genomic
stability. Strikingly, we found that attenuation of replication strongly
suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells.
Our findings thus provide a direct demonstration that R-loop formation impairs
DNA replication and that this is responsible for the deleterious effects of R
loops on genome stability from bacteria to humans. We show that the time required to transcribe human genes larger than 800 kb
spans more than one complete cell cycle, while their transcription speed equals
that of smaller genes. Independently of their expression status, we find the
long genes to replicate late. Regions of concomitant transcription and
replication in late S phase exhibit DNA break hot spots known as common fragile
sites (CFSs). This CFS instability depends on the expression of the underlying
long genes. We show that RNA:DNA hybrids (R-loops) form at sites of
transcription/replication collisions and that RNase H1 functions to suppress CFS
instability. In summary, our results show that, on the longest human genes,
collisions of the transcription machinery with a replication fork are
inevitable, creating R-loops and consequent CFS formation. Functional
replication machinery needs to be involved in the resolution of conflicts
between transcription and replication machineries to ensure genomic stability. DNA and RNA polymerases clash along the genome as they compete for the same DNA
template. Cells have evolved specialized strategies to prevent and resolve
replication and transcription interference. Here, we review the topology and
architecture at sites of replication fork clashes with transcription bubbles as
well as the regulatory circuits that control replication fork passage across
transcribed genes. In the case of RNA polymerase II-transcribed genes,
cotranscriptional processes such as mRNA maturation, splicing, and export
influence the integrity of replication forks and transcribed loci. Fork passage
likely contributes to reset the epigenetic landscape, influencing gene
expression and transcriptional memory. When any of these processes are not
properly coordinated, aberrant outcomes such as fork reversal and R-loop
formation arise and trigger unscheduled recombinogenic events and genome
rearrangements. The evolutionary implications of such conflicts on genome
dynamics and their potential impact on oncogenic stress are discussed. In human mitochondria the transcription machinery generates the RNA primers
needed for initiation of DNA replication. A critical feature of the
leading-strand origin of mitochondrial DNA replication is a CG-rich element
denoted conserved sequence block II (CSB II). During transcription of CSB II, a
G-quadruplex structure forms in the nascent RNA, which stimulates transcription
termination and primer formation. Previous studies have shown that the newly
synthesized primers form a stable and persistent RNA-DNA hybrid, a R-loop, near
the leading-strand origin of DNA replication. We here demonstrate that the
unusual behavior of the RNA primer is explained by the formation of a stable
G-quadruplex structure, involving the CSB II region in both the nascent RNA and
the non-template DNA strand. Based on our data, we suggest that G-quadruplex
formation between nascent RNA and the non-template DNA strand may be a regulated
event, which decides the fate of RNA primers and ultimately the rate of
initiation of DNA synthesis in human mitochondria. R-loop is a type of three-stranded nucleic acid structure that is made up of an
RNA:DNA hybrid, formed due to failing separation of a nascent RNA molecule with
transcripting template in transcription or by the re-annealing of RNA molecule
with one of the two strands in a double stranded DNA molecule, along with the
single stranded DNA, which is either the non-template strand in the
transcription bubble or the RNA substituted DNA strand. Formation of R-loops can
occur when transcription goes through a genomic DNA region having a tract of G
bases in the non-template strand in the transcription bubble or through a type
of triplet microsatellite DNA sequences that are known to be associated with
certain human diseases. The negative supercoiling forces accumulated in the
transcription bubble, and the misprocessing of RNA precursors, as well as the
delayed utilizations and transportations of RNA molecules to cytoplasm promote R
loop formation. Many studies show that cells can manage R loop formation with
efficiency, and can also process the R-loops already formed in the cell, and by
which, the bad effects of R-loops on DNA replication, gene mutation and
homologous recombination can be regulated. In this review, we summarize the
formation and the impacts of R-loops on DNA replication, mutation rates and the
frequencies of homologous recombination, and also discusse the possible role of
the R-loop induced DNA replication in mediating trinucleotide repeat expansions
as seen with those frequently associated with human neuromuscular degenerative
diseases. In bacterial cells, bidirectional replication of the circular chromosome is
initiated from a single origin (oriC) and terminates in an antipodal terminus
region such that movement of the pair of replication forks is largely
codirectional with transcription. The terminus region is flanked by discrete Ter
sequences that act as polar, or direction-dependent, arrest sites for fork
progression. Alternative oriC-independent modes of replication initiation are
possible, one of which is constitutive stable DNA replication (cSDR) from
transcription-associated RNA-DNA hybrids or R-loops. Here, I discuss the
distinctive attributes of fork progression and termination associated with
different modes of bacterial replication initiation. Two hypothetical models are
proposed: that head-on collisions between pairs of replication forks, which are
a feature of replication termination in all kingdoms of life, provoke bilateral
fork reversal reactions; and that cSDR is characterized by existence of distinct
subpopulations in bacterial cultures and a widespread distribution of origins in
the genome, each with a small firing potential. Since R-loops are known to exist
in eukaryotic cells and to inflict genome damage in G1 phase, it is possible
that cSDR-like events promote aberrant replication initiation even in
eukaryotes. The uticipated widespread occurrence of stable hybrid DNA/RNA structures
(R-loops) in human cells and the increasing evidence of their involvement in
several human maligcies have invigorated the research on R-loop biology in
recent years. Here we propose that physiological R-loop formation at CpG island
promoters can contribute to DNA replication origin specification at these
regions, the most efficient replication initiation sites in mammalian cells.
Quite likely, this occurs by the strand-displacement reaction activating the
formation of G-quadruplex structures that target the origin recognition complex
(ORC) in the single-stranded conformation. In agreement with this, we found that
R-loops co-localize with the ORC within the same CpG island region in a
significant fraction of these efficient replication origins, precisely at the
position displaying the highest density of G4 motifs. This scenario builds on
the connection between transcription and replication in human cells and suggests
that R-loop dysregulation at CpG island promoter-origins might contribute to the
phenotype of DNA replication abnormalities and loss of genome integrity detected
in cancer cells. |
Which two catechol-O-methyl transferase (COMT) inhibitors can be used for treatment of Parkinson disease? | Tolcapone (central and peripheral) and entacapone (peripheral) are catechol-O-methyl transferase inhibitors that are used for treatment of Parkinson disease. | Flurodopa (FDOPA) is an analogue of L-di-hydroxyphenylalanine (L-dopa) used to
assess the nigrostriatal dopamine system in vivo with positron emission
tomography (PET). However, FDOPA/PET quantitation is complicated by the presence
of the 3-O-methyl-FDOPA (3OMFD) fraction in brain and plasma. Pretreatment with
entacapone (OR-611), a peripheral catechol O-methyl-transferase (COMT)
inhibitor, greatly reduces the plasma 3OMFD fraction and provides an ideal
situation to evaluate the contribution of the plasma 3OMFD fraction in several
kinetic models of FDOPA uptake. We performed FDOPA/PET with and without the
OR-611 preadministration in six Parkinson's disease (PD) patients. We measured
the time-course of the plasma FDOPA and 3OMFD fractions using high-pressure
liquid chromatography (HPLC). We calculated striato-occipital ratios (SOR), and
estimated the striatal FDOPA uptake rate constant graphically using the plasma
FDOPA and occipital tissue time activity curves (KiFD and KiOCC, respectively).
We also estimated striatal dopa decarboxylase (DDC) activity (k3D) using a model
incorporating independent measurements of 3OMFD transport kinetic rate
constants. With the preadministration of OR-611, the pharmacological efficiency
in plasma was prolonged significantly (21.1-37.7%; p < 0.01). We also observed
significant mean elevations in SOR and KiOCC by 21.8 and 53.5%, respectively (p
< 0.05). KiFD and k3D did not show significant change. We conclude that OR-611
prolongs the circulation time of FDOPA in the plasma but does not alter rate
constants for striatal FDOPA uptake or decarboxylation. In order to study whether the membrane hyperpolarization and firing inhibition
caused by dopamine and levodopa on rat midbrain dopamine cells are affected by
the inhibition of brain catechol-O-methyl-transferase (COMT), intracellular
electrophysiological recordings were made from these neurons maintained in
vitro. Here we report that a treatment of the cerebral tissue with tolcapone, a
central and peripheral inhibitor of COMT, does not change the membrane responses
of midbrain dopamine neurons to dopamine and levodopa. The lack of modification
of the dopaminergic effects by tolcapone suggests that the pharmacological
inhibition of intracerebral COMT does not have detectable action on dopamine
neurotransmission. Therefore, the therapeutic action of tolcapone in Parkinson's
disease, might be dependent on the reduction of COMT activity in the
extracerebral tissue. Two inhibitors of catechol-O-methyl transferase (COMT), tolcapone and
entacapone, have recently been introduced as adjuncts to levodopa in the
treatment of Parkinson's disease patients. Both have been shown to provide PD
patients with increased "on" time, decreased "off" time, and improved motor
scores. There are, however, a number of practical issues that must be considered
in order to achieve maximal benefits with this class of agent. They include
dosing and administration, efficacy, adverse events, and patient education. In
general, these agents are easy to administer and well tolerated. Both the
benefits and the principal side effects of treatment are related to increased
dopaminergic activity. Patients must be advised of possible side effects so that
they can be reported in a timely manner. Physicians must appreciate that
dopaminergic side effects, such as dyskinesia, should be controlled by adjusting
the dose of levodopa and not the COMT inhibitor. Explosive diarrhea has been
reported with tolcapone and usually necessitates discontinuing the drug.
Tolcapone must also be monitored for possible liver dysfunction. This has not
been reported with entacapone, and no monitoring is required. Metabolites of
tolcapone and entacapone may cause discoloration of the urine. This is harmless
but patients should be advised that this may occur. Catechol-O-methyl transferase (COMT) inhibitors block the peripheral metabolism
of levodopa, increase its plasma half-life, and enhance its brain availability.
Two COMT inhibitors, tolcapone and entacapone, have recently been made available
as adjunctive agents to levodopa. In PD patients with motor fluctuations, they
have been shown to increase "on" time and reduce "off" time. In patients with
more advanced disease, they provide similar benefits, but patients tend to
experience less overall benefit and a greater likelihood of developing
dopaminergic adverse events. Accordingly, closer monitoring is required. In
stable patients who have not yet developed motor complications, there are
preliminary data suggesting that they experience improvements in motor function
and in activities of daily living. Finally, there are theoretical reasons to
consider administering a COMT inhibitor to patients from the onset of levodopa
therapy in order to reduce the likelihood that motor complications will develop.
COMT inhibitors are easy to administer, do not require titration, and are
generally well tolerated particularly in patients with relatively mild disease.
Adverse events are primarily dopaminergic and can usually be controlled by
levodopa dose adjustments. COMT inhibitors have thus proven to be a useful
addition to the therapeutic armamentarium of PD. We present a patient who suffered from sleep attacks after starting entacapone
in addition to levodopa. Entacapone, a catechol-O-methyl transferase inhibitor,
alters the pharmacokinetics of levodopa, leading to increase of levodopa
concentration in plasma and brain. This mechanism is suspected to be involved in
the pathophysiology of sleep attacks in this case. The catechol-O-methyl transferase inhibitor entacapone is given in combination
with levodopa/dopa decarboxylase inhibitor for Parkinson's disease (PD) patients
experiencing end-of-dose wearing-off. This 4-week post-marketing surveillance
study was undertaken to assess patients' responses to levodopa combined with
entacapone in a real clinical practice setting. Overall, 466 patients with
idiopathic PD treated with levodopa and experiencing symptoms of wearing-off
were recruited. Both physicians and patients recorded the response to therapy,
including improvements and side-effects. Following initiation of entacapone
treatment, the average daily levodopa dose was reduced from 510 to 453 mg.
Physician assessment of entacapone efficacy was judged to be "very good" or
"good" in 77.6% of the patients, and tolerability was considered to be "very
good" or "good" in 92.4% of patients, with only 12 patients (2.6%) withdrawing
from the study. Compared with baseline, there was a decrease in the mean
duration of daily 'off' time from 3.0 to 1.3 h per day during the treatment
period. Adverse events were in line with those previously reported, with
diarrhoea being the most frequent event. The percentage of patients suffering
from dyskinesia decreased from 46 to 34%, and of those patients still suffering
from dyskinesia, the average daily duration of dyskinesia was reduced from 2.2
to 1.7 h. The use of adjunct dopamine agonists decreased from 67 to 59%. At
study end, the percentage of patients who rated their quality of life (QoL) as
"very good" or "good" increased from 12.1 to 51.7% and the percentage of
patients who rated their QoL as "bad" or "very bad" decreased from 40 to 10.7%.
In summary, the results of this survey conducted in real clinical practice
support the findings of previous clinical trials demonstrating the efficacy and
tolerability of entacapone, as well as the benefits of improved QoL, for
patients achieved with entacapone. We investigated whether administration of the catechol-O-methyl transferase
(COMT) inhibitor entacapone, at doses of 200 mg and 400 mg, alters the
pharmacokinetics of apomorphine in Parkinson's disease patients experiencing
severe motor fluctuations. In addition, the pharmacodynamics and safety of
entacapone and apomorphine coadministration in these patients were examined. The
study followed a three-sequence, three-period, crossover design. Patients were
randomly assigned to one of three sequences that included single oral doses of
entacapone 200 mg, entacapone 400 mg, and placebo in a predefined order. On 3
separate test days, study treatment was administered before apomorphine. The
study evaluations (pharmacokinetics, tapping test, and dyskinesia evaluation
[Abnormal Involuntary Movements Scale - AIMS]) were performed on these days.
Furthermore, Unified Parkinson Disease Rating Scale (UPDRS) scores were
evaluated at baseline and study end. Pharmacokinetic parameters for apomorphine
(C(max), AUC, t(max), t(1/2)) were unchanged by the administration of
entacapone, and changes in both the tapping test and AIMS score were similar
with all treatments (entacapone 200 mg, entacapone 400 mg, and placebo). There
was no significant difference in mean total UPDRS scores between baseline and
study end. The administration of entacapone did not change the pharmacokinetic
or pharmacodynamic effects of apomorphine in these patients or prolong the
clinical effect of apomorphine. Thus, apomorphine may be safely administered to
patients receiving therapy with levodopa and entacapone, providing a useful
addition to treatment for patients with advanced Parkinson's disease. The year of 2007 was a turning point of the treatment of Parkinson's disease
(PD) in Japan. Severe adverse effects of dopamine agonists including valvular
heart disease induced by ergots and sudden onset of sleep attacks induced by
non-ergots, were disclosed, and treatments with agonists were reassessed. Good
news were marketing of ropinirole, a new non-ergot agonist, in December 2006 and
entacapone, the first catechol-O-methyl transferase (COMT) inhibitor in Japan in
April 2007. Having faced these new situations, Japanese Neurological Association
has started revising "the Guideline 2002 for the treatment of Parkinson's
disease". Clinical trials of translational gene therapy for Parkinson's disease
with adeno-associated virus (AAV) vector are now going on in four approaches:
restoring dopamine synthetic capacity, protecting against cell death with
trophic factors, interfering with the aberrant protein aggregation, and
converting the subthalamic nucleus into an inhibitory, rather than an
excitatory, structure. In Japan, gene delivery of the dopamine synthesizing
enzyme aromatic amino acid decarboxylase (AADC) to the striatum of PD patients
is going on in Jichi Medical University. New findings of the causative genes,
environmental factors and molecular mechanism of PD have provided with new tools
for developing new treatments. The big success of induction of induced
pluripotent stem (iPS) cells from fibroblast has given an impact on cell therapy
research of PD. Levodopa is the most efficacious agent for the treatment of motor features of
Parkinson's disease but its chronic use is associated with the development of
motor complications. Mounting evidence indicates the short half-life of levodopa
and resultant pulsatile stimulation of striatal dopamine receptors leads to
wearing off, motor fluctuations and dyskinesias. Longer acting dopaminergic
agents, such as dopamine agonists, are less likely to cause motor fluctuations
and dyskinesias but are not as efficacious for control of motor symptoms.
Therefore, there is interest in exploring ways to deliver levodopa in a more
continuous fashion, in an effort to maintain benefit through the day and reduce
the development of motor fluctuations and dyskinesias. A dopa decarboxylase
inhibitor (DDCI), such as carbidopa or benserazide, is administered with
levodopa to attenuate its peripheral conversion to dopamine, reduce nausea and
increase central bioavailability. When levodopa is administered with a DDCI, its
main route of peripheral metabolism is via catechol-O-methyl transferase (COMT).
A COMT inhibitor can be added to the combination of levodopa and a DDCI to
further extend the levodopa peripheral half-life and increase central
bioavailability. Stalevo is a combination tablet comprised of levodopa,
carbidopa, and the COMT inhibitor entacapone. It is available in fixed-dose
combinations of levodopa/carbidopa/entacapone, 50/12.5/200, 75/18.75/200,
100/25/200, 125/31.25/200, 150/37.5/200 and 200/50/200 mg. Stalevo is currently
approved for use in Parkinson's disease patients with end-of-dose wearing off. OBJECTIVES: Entacapone is a highly potent, reversible, peripherally acting
catechol-O-methyl transferase (COMT) inhibitor that is used as an adjunct to
L-dopa in the treatment of patients with Parkinson disease (PD). Nevertheless,
the consequence of the long-lasting inhibition of COMT by entacapone has never
been investigated. We assessed the variation of the soluble red blood cell
(S-RBC)-COMT activity after 3 months of chronic treatment by entacapone.
METHODS: Twelve consecutive white PD patients (3 women and 9 men; mean age, 65.7
± 2.4 years) with L-dopa-related motor fluctuations were assessed. Entacapone
200 mg was given in combination with each scheduled L-dopa/dopa decarboxylase
inhibitor dose (range, 3-5 doses daily). The S-RBC-COMT activity was determined
both before entacapone administration (baseline) and twice, respectively, after
1 and 3 months treatment with entacapone, that is, on morning, after at least a
12-hour withdrawal of entacapone and L-dopa and before the following first daily
administration.
RESULTS: Mean baseline S-RBC-COMT activity was 0.72 ± 0.09 pmol/min per
milligram (range, 0.30-1.29 pmol/min per milligram) of protein. After 3 months,
the level increased significantly in all PD patients from 0.72 ± 0.09 pmol/min
per milligram (range, 0.30-1.29 pmol/min per milligram) to 1.19 ± 0.13 pmol/min
per milligram (range, 0.58-2.14 pmol/min per milligram) of protein (P < 0.01),
which corresponds to a mean increase of 72.9 ± 9.2% (range, 24%-146%).
CONCLUSIONS: Our findings suggest that a long-lasting inhibition of the COMT may
limit the efficacy of entacapone by development of a tolerance. Moreover, one
may assume that an abrupt withdrawal of the treatment will be followed by a
dramatic worsening of motor disability. |
What are the structures formed when keratin molecules come together? | Keratins form the intermediate filaments of the cytoskeleton and provide scaffold structures within cells. | Desmosomes are cell-cell adhesion structures that integrate cytoskeletal
networks. In addition to binding intermediate filaments, the desmosomal protein
desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this
paper, we identify a specific subset of centrosomal proteins that are recruited
to the cell cortex by DP upon epidermal differentiation. These include Lis1 and
Ndel1, which are centrosomal proteins that regulate microtubule organization and
anchoring in other cell types. This recruitment was mediated by a region of DP
specific to a single isoform, DPI. Furthermore, we demonstrate that the
epidermal-specific loss of Lis1 results in dramatic defects in microtubule
reorganization. Lis1 ablation also causes desmosomal defects, characterized by
decreased levels of desmosomal components, decreased attachment of keratin
filaments, and increased turnover of desmosomal proteins at the cell cortex.
This contributes to loss of epidermal barrier activity, resulting in completely
penetrant perinatal lethality. This work reveals essential desmosome-associated
components that control cortical microtubule organization and unexpected roles
for centrosomal proteins in epidermal function. Re-modeling of epithelial tissues requires that the cells in the tissue
rearrange their adhesive contacts in order to allow cells to migrate relative to
neighboring cells. Desmosomes are prominent adhesive structures found in a
variety of epithelial tissues that are believed to inhibit cell migration and
invasion. Mechanisms regulating desmosome assembly and stability in migrating
cells are largely unknown. In this study we established a cell culture model to
examine the fate of desmosomal components during scratch wound migration.
Desmosomes are rapidly assembled between epithelial cells at the lateral edges
of migrating cells and structures are transported in a retrograde fashion while
the structures become larger and mature. Desmosome assembly and dynamics in this
system are dependent on the actin cytoskeleton prior to being associated with
the keratin intermediate filament cytoskeleton. These studies extend our
understanding of desmosome assembly and provide a system to examine desmosome
assembly and dynamics during epithelial cell migration. An intact keratin 5/keratin 14 intermediate filament cytoskeleton is vital for
the integrity of basal keratinocytes and for the development and maintece of
epidermal structures. In patients with epidermolysis bullosa simplex
Dowling-Meara (EBS-DM), heterozygous mutations in the keratin 14 gene in
keratinocytes cause a cytoskeletal collapse leading to fragile cells susceptible
to cellular stress. The primary aim of this work was to extend analysis of
differentially expressed genes in an EBS-DM model cell line to obtain insights
into the molecular consequences resulting from the keratin 14 mutation. In a
first step, suppression subtractive hybridization (SSH), a powerful technology
to enrich for differentially expressed genes, was used to identify genes whose
up-regulation may be a direct or indirect result of the keratin 14 mutation,
R125P. We discovered 55 candidate genes (SSH genes) that were further analysed
by RTq-PCR. Of the 55 SSH genes, 14 (25.45%) were found to be congruently
up-regulated. Bioinformatic analysis revealed significant enrichment of genes
regulating epidermal development, migration, apoptosis and wound healing. Maintaining proper cell-cell adhesion in the intestine is essential for tissue
homeostasis and barrier function. This adhesion is thought to be mediated by
cell adhesion structures, including tight junctions, adherens junctions, and
desmosomes, which concentrate in the apical junctional region. While clear roles
for adherens and tight junctions have been established in simple epithelia, the
function of desmosomes has not been addressed. In stratified epithelia,
desmosomes impart mechanical strength to tissues by organizing and anchoring the
keratin filament network. In this paper, we report that the desmosomal protein
desmoplakin (DP) is not essential for cell adhesion in the intestinal
epithelium. Surprisingly, when DP is lacking, keratin filament localization is
also unperturbed, although keratin filaments no longer anchor at desmosomes.
Unexpectedly, DP is important for proper microvillus structure. Our study
highlights the tissue-specific functions of desmosomes and reveals that the
canonical functions for these structures are not conserved in simple epithelium. We have investigated depth-resolved cellular structures of unmodified fresh
human scalp hairs with ultrahigh-resolution full-field optical coherence
tomography (FF-OCT). The Linnik-type white light interference microscope has
been home-implemented to observe the micro-internal layers of human hairs in
their natural environment. In hair shafts, FF-OCT has qualitatively revealed the
cellular hair compartments of cuticle and cortex layers involved in keratin
filaments and melanin granules. No significant difference between black and
white hair shafts was observed except for absence of only the melanin granules
in the white hair, reflecting that the density of the melanin granules directly
affects the hair color. Anatomical description of plucked hair bulbs was also
obtained with the FF-OCT in three-dimensions. We expect this approach will be
useful for evaluating cellular alteration of natural hairs on cosmetic
assessment or diagnosis of hair diseases. FasR stimulation by Fas ligand leads to rapid formation of FasR microaggregates,
which become signaling protein oligomerization transduction structures (SPOTS),
through interactions with actin and ezrin, a structural step that triggers
death-inducing signaling complex formation, in association with procaspase-8
activation. In some cells, designated as type I, caspase 8 directly activates
effector caspases, whereas in others, known as type II, the caspase-mediated
death signaling is amplified through mitochondria. Keratins are the intermediate
filament (IF) proteins of epithelial cells, expressed as pairs in a
lineage/differentiation manner. Hepatocyte IFs are made solely of keratins 8/18
(K8/K18), the hallmark of all simple epithelia. We have shown recently that in
comparison to type II wild-type (WT) mouse hepatocytes, the absence of K8/K18
IFs in K8-null hepatocytes leads to more efficient FasR-mediated apoptosis, in
link with a type II/type I-like switch in FasR-death signaling. Here, we
demonstrate that the apoptotic process occurring in type I-like K8-null
hepatocytes is associated with accelerated SPOTS elaboration at surface
membrane, along with manifestation of FasR cap formation and internalization. In
addition, the lipid raft organization is altered in K8-null hepatocytes. While
lipid raft inhibition impairs SPOTS formation in both WT and K8-null
hepatocytes, the absence of K8/K18 IFs in the latter sensitizes SPOTS to actin
de-polymerization, and perturbs ezrin compartmentalization. Overall, the results
indicate that the K8/K18 IF loss in hepatocytes alters the initial FasR
activation steps through perturbation of ezrin/actin interplay and lipid raft
organization, which leads to a type II/type I switch in FasR-death signaling. Keratin is a protein in the intermediate filament family and the key component
of hair, nail, and skin. Here we report a bottom-up atomistic model of the
keratin dimer, using the complete human keratin type k35 and k85 amino acid
sequence. A detailed analysis of geometric and mechanical properties through
full-atomistic simulation with validation against experimental results is
presented. We introduce disulfide cross-links in a keratin tetramer and compare
the mechanical behavior of the disulfide bonded systems with a system without
disulfide bonds. Disulfide bond results in a higher strength (20% increase) and
toughness (49% increase), but the system loses α-helical structures under
loading, suggesting that disulfide bonds play a significant role in achieving
the characteristic mechanical properties of trichocyte α-keratin. Our study
provides general insight into the effect of disulfide cross-link on mechanical
properties. Moreover, the availability of an atomistic model of this protein
opens the possibility to study the mechanical properties of hair fibrils and
other fibers from a bottom-up perspective. BACKGROUND: The stratum corneum (SC) is the outermost region of the epidermis
and plays key roles in cutaneous barrier function in mammals. The SC is composed
of 'bricks', represented by flattened, protein-enriched corneocytes, and
'mortar', represented by intercellular lipid-enriched layers. As a result of
this 'bricks and mortar' structure, the SC can be considered as a 'rampart' that
encloses water and solutes essential for physiological homeostasis and that
protects mammals from physical, chemical and biological assaults.
STRUCTURES AND FUNCTIONS: The corneocyte cytoskeleton contains tight bundles of
keratin intermediate filaments aggregated with filaggrin monomers, which are
subsequently degraded into natural moisturizing compounds by various proteases,
including caspase 14. A cornified cell envelope is formed on the inner surface
of the corneocyte plasma membrane by transglutaminase-catalysed cross-linking of
involucrin and loricrin. Ceramides form a lipid envelope by covalently binding
to the cornified cell envelope, and extracellular lamellar lipids play an
important role in permeability barrier function. Corneodesmosomes are the main
adhesive structures in the SC and are degraded by certain serine proteases, such
as kallikreins, during desquamation.
CLINICAL RELEVANCE: The roles of the different SC components, including the
structural proteins in corneocytes, extracellular lipids and some proteins
associated with lipid metabolism, have been investigated in genetically
engineered mice and in naturally occurring hereditary skin diseases, such as
ichthyosis, ichthyosis syndrome and atopic dermatitis in humans, cattle and
dogs. BACKGROUND: In breast cancer the development of metastasis is a major turning
point in the treatment and outcome of the disease. Throughout tumour
development, and especially in the development of metastasis, epithelial
mesenchymal transition takes place. During this transformation into a
mesenchymal phenotype, the tumour cells undergo a series of structural changes.
The loss of structural integrity and adoption of mesenchymal filaments enables
cells to detach from the epithelial cell layer and metastasise. Keratins form
the intermediate filaments of the cytoskeleton and provide scaffold structures
within cells. During cancer progression the intermediate filaments are
reorganised, and dramatic changes are seen in their protein components. Keratins
K8, K18, K19 and vimentin are intermediate filament proteins with altered
expression profiles during tumour development.
METHOD: We have used in vivo and in vitro models to analyse changes in
intermediate filament proteins. Antibody-based methods were used to study K8
levels and proteomic analysis to profile the protein content of metastatic
breast cancer cell variants.
RESULTS: K8 expression declines as human breast tumours progress into an
invasive phenotype. Analysis of IF proteins indicated altered expression
profiles of K8, K18, K19 and vimentin, with K8, K18, K19 expressed in high
levels in the T47D and MCF-7 cell lines, whereas the highly metastatic cell
lines expressed lower levels of K8 and K18 and no detectable K19. Vimentin
showed reverse expression profile with T47D and MCF-7 cells having no detectable
vimentin expression whereas the highly metastatic MDA-MB-231 and MDA-MB-436
showed high levels. Analysis of acetylation status using specific antibodies
suggested acetylation occurred within the central coiled domain in the MCF-7 and
T47D cells. Inhibition of tumour growth by tissue factor (TF) shRNA resulted in
a dramatic re-elevation of expression of K8 in xenographs of the highly
metastatic MDA-MB-436 line.
CONCLUSION: Intermediate filament expression alters during epithelial
mesenchymal transition. Identified post translational modifications may play a
role in alterations seen in the organisation, solubility and stability of these
filaments. Epithelial mesenchymal transition can be reversed and an epithelial
phenotype re-established. |
Which is the prognostic impact of hypothyroidism in patients with acute myocardial infarction? | Thyroid dysfunction, particularly low T3 syndrome, is a strong predictor of short-term and long-term poor prognoses in patients with acute myocardial infarctions. | PURPOSE: The thyroid hormone system may be downregulated temporarily in patients
who are severely ill. This "euthyroid sick syndrome" may be an adaptive response
to conserve energy. However, thyroid hormone also has beneficial effects on the
cardiovascular system, such as improving cardiac function, reducing systemic
vascular resistance, and lowering serum cholesterol levels. We investigated
whether thyroid hormone levels obtained at the time of myocardial infarction are
associated with subsequent mortality.
PATIENTS AND METHODS: Serum levels of thyroid hormones (triiodothyronine [T3],
reverse T3, free thyroxine [T4], and thyroid-stimulating hormone) were measured
in 331 consecutive patients with acute myocardial infarction (mean age [+/- SD],
68 +/- 12 years), from samples obtained at the time of admission.
RESULTS: Fifty-three patients (16%) died within 1 year. Ten percent (16 of 165)
of patients with reverse T3 levels (an inactive metabolite) >0.41 nmol/L (the
median value) died within the first week after myocardial infarction, compared
with none of the 166 patients with lower levels (P <0.0004). After 1 year, the
corresponding figures were 24% (40 of 165) versus 7.8% (13 of 166; P <0.0001).
Reverse T3 levels >0.41 nmol/L were associated with an increased risk of 1-year
mortality (hazard ratio = 3.0; 95% confidence interval: 1.4 to 6.3; P = 0.005),
independent of age, previous myocardial infarction, prior angina, heart failure,
serum creatinine level, and peak serum creatine kinase-MB fraction levels.
CONCLUSION: Determination of reverse T3 levels may be a valuable and simple aid
to improve identification of patients with myocardial infarction who are at high
risk of subsequent mortality. The purpose of this study was to investigate whether thyroid hormone levels have
any predictive value for mortality in patients presenting to the emergency
department with acute myocardial infarction (AMI). Three groups of patients
admitted to the emergency department within the 11-month study period were
considered eligible: 95 patients with chest pain and proven AMI, 26 patients
with chest pain and no AMI, and 114 patients who served as controls with no
evidence of any major disease. Cardiac enzymes and the following thyroid
hormones were analyzed and compared between groups, regarding effects of
historical and demographic factors: thyrotrophin, free triiodothyronine (FT3),
total triiodothyronine (TT3), free thyroxine (FT4), and total thyroxine (TT4).
Sixteen patients with AMI (16.8%) died within the study period. Troponin T and
creatine kinase-B with an M-type subunit levels were significantly higher in the
nonsurvivors when compared with survivors. Survivors in the AMI group had higher
TT3, TT4, and lower FT4 levels, while the nonsurvivors in the AMI group had
higher thyrotrophin and lower TT3, FT3 and FT4 levels than controls. In the AMI
group, the nonsurvivors had lower TT3 and FT3 levels than the survivors. A
history of diabetes mellitus and/or angina, TT3, or FT3 was an independent
predictor of mortality. TT3 and FT3 appear to be independent prognostic factors
in patients with AMI. BACKGROUND: Overt thyroid dysfunction, hypothyroidism in particular, may lead to
coronary artery disease (CAD). Whether more subtle anomalies of thyroid hormone
metabolism influence the progression of CAD remains a matter of speculation.
HYPOTHESIS: The occurrence of CAD and long-term prognosis in patients without a
history of either primary thyroid disease, myocardial infarction, or chronic
heart failure is related to serum levels of biologically active free
triiodothyronine (fT3).
METHODS: The cohort consisted of 1047 clinically and biochemically euthyroid
patients (median age 65.6 y and 69% male) who underwent coronary angiography in
our institute for suspected CAD.
RESULTS: Lower fT3 levels were predictive of both single-vessel (p = 0.012) and
multivessel (p = 0.009) CAD. Through a multivariate logistic regression
analysis, fT3 was still linked to the presence of CAD (hazard ratio [HR]: 0.48,
95% confidence interval [CI]: 0.34-0.68, p < 0.001). After a mean follow-up of
31 months, the survival rate was 95% and total mortality (log-rank 6.75, p =
0.009), as well as cardiac mortality (log-rank 8.26, p = 0.004), was greater
among patients with low T3 (fT3 < 2.10 pg/mL) syndrome. At subsequent
multivariate Cox regression analysis, the association between low T3 syndrome
and survival was maintained (total mortality HR: 1.80, 95% CI: 1.05-3.10, p =
0.034; cardiac mortality HR: 2.58, 95% CI: 1.13-5.93, p = 0.025).
CONCLUSIONS: In this selected population, fT3 levels were inversely correlated
to the presence of CAD and low T3 syndrome conferred an adverse prognosis, even
after adjusting for traditional coronary risk factors. OBJECTIVE: To investigate the association between low free triiodothyronine
(fT3) levels and the severity and prognosis of patients with acute myocardial
infarction.
METHODS: A total of 501 patients with acute myocardial infarctions were enrolled
in our study. The circulating levels of thyroid hormones and clinical parameters
were assayed. The patients were categorized into either the low fT3 group or the
normal fT3 group according to the fT3 level on admission. All patients underwent
a follow-up for 10±2 months for mortality from any cause and the occurrence of
any adverse major cardiac events (MACE).
RESULTS: There were 171 patients in the low fT3 group (fT3<3.5 pmol/L) and 330
patients in the normal fT3 group (≥3.5 pmol/L). During the follow-up period, 33
patients died (6.6%) and the overall survival rates were 86.0% and 97.3% in
patients with a low fT3 level and a normal fT3 level, respectively. The rates of
MACE were 66.7% and 45.5% in the patients with and those without low fT3 levels,
respectively. Using a multivariable Cox proportional hazards model, the fT3
level was found to be the most important predictor of cumulative death and MACE
(hazard ratio [HR] for death: 0.142, p<0.001 and HR for major adverse cardiac
events: 0.748, p=0.007). A Kaplan-Meier analysis revealed that those patients
with low fT3 levels had higher rates of MACE and death.
CONCLUSION: A low fT3 level, a common phenomenon in patients with acute
myocardial infarctions, is a strong predictor of short-term and long-term poor
prognoses in patients with acute myocardial infarctions. BACKGROUND: Concomitant thyroid and heart disease are frequently encountered in
clinical practice. There are many studies evaluating thyroid function in acute
and critical conditions. Information on thyroid dysfunction in ST-segment
elevation myocardial infarction (STEMI) is limited; its correlation with short
and long-term outcome is not fully known.
METHODS: Four hundred and fifty seven patients diagnosed with STEMI in our
emergency department were included in the study. Patients were divided into two
groups: patients with normal thyroid function (euthyroid) and patients with
thyroid dysfunction. STEMI was diagnosed with 12 derivation surface
electrocardiogram. Thyroid hormone levels (TSH, free T3 and free T4) were
measured. Patients with other acute coronary syndromes and endocrine pathologies
except diabetes mellitus were excluded. Two patient groups were compared in
terms of in-hospital and long-term outcome.
RESULTS: Out of 457, 72 (15%) patients with thyroid dysfunction were detected.
The other patients were euthyroid and constituted the control group. In-hospital
cardiogenic shock (15% vs. 3% in the control group; p < 0.01) and death (7% vs.
1% in the control group; p < 0.01) were more frequently observed in the thyroid
dysfunction group. In the subgroup analysis, it was observed that patients with
sick euthyroid syndrome have the poorest outcome. Other markers for poor outcome
were anemia and renal failure.
CONCLUSIONS: Thyroid dysfunction, particularly sick euthyroid syndrome, was
found to be related to in-hospital and long term mortality in patients with
STEMI undergoing primary percutaneous intervention. |
Which are the main features of CREST and other ALS-linked proteins? | CREST and certain other ALS-linked proteins share several features implicated in ALS pathogenesis, namely the ability to aggregate, be recruited to stress granules and alter paraspeckle integrity. | INTRODUCTION: Recently it has been shown in animal models of amyotrophic lateral
sclerosis (ALS) that stem cells significantly slow the progression of the
disease and prolong survival. We have evaluated the feasibility and safety of a
method of intraspinal cord implantation of autologous mesenchymal stem cells
(MSCs) in a few well-monitored patients with ALS.
METHOD: Bone marrow collection was performed according to the standard procedure
by aspiration from the posterior iliac crest. Ex vivo expansion of mesenchymal
stem cells was induced according to Pittenger's protocol. The cells were
suspended in 2 ml of autologous cerebrospinal fluid and transplanted into the
spinal cord by a micrometric pump injector.
RESULTS: No patient manifested major adverse events such as respiratory failure
or death. Minor adverse events were intercostal pain irradiation (4 patients)
which was reversible after a mean period of three days after surgery, and leg
sensory dysesthesia (5 patients) which was reversible after a mean period of six
weeks after surgery. No modification of the spinal cord volume or other signs of
abnormal cell proliferation were observed.
CONCLUSIONS: Our results appear to demonstrate that the procedures of ex vivo
expansion of autologous mesenchymal stem cells and of transplantation into the
spinal cord of humans are safe and well tolerated by ALS patients. OBJECTIVES: Our study was aimed to evaluate the feasibility and safety of
intraspinal cord implantation of autologous mesenchymal stem cells (MSCs) in a
few well-monitored amyotrophic lateral sclerosis (ALS) patients.
METHODS: Seven patients affected by definite ALS were enrolled in the study and
two patients were treated for compassionate use and monitored for at least 3
years. Bone marrow was collected from the posterior iliac crest according to the
standard procedure and MSCs were expanded ex vivo according to Pittenger's
protocol. The cells were suspended in 2 ml autologous cerebrospinal fluid and
transplanted into the spinal cord by a micrometric pump injector.
RESULTS: The in vitro expanded MSCs did not show any bacterial o fungal
contamination, hemopoietic cell contamination, chromosomic alterations and early
cellular senescence. No patient manifested major adverse events such as
respiratory failure or death. Minor adverse events were intercostal pain
irradiation and leg sensory dysesthesia, both reversible after a mean period of
6 weeks. No modification of the spinal cord volume or other signs of abnormal
cell proliferation were observed. A significant slowing down of the linear
decline of the forced vital capacity was evident in four patients 36 months
after MSCs transplantation.
CONCLUSIONS: Our results demonstrate that direct injection of autologous
expanded MSCs into the spinal cord of ALS patients is safe, with no significant
acute or late toxicity, and well tolerated. The clinical results seem to be
encouraging. Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial
amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding
protein but how PFN1 mutations cause ALS is unknown. The budding yeast,
Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked
profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability
and failure to restore growth to profilin mutant cells, without exhibiting
gain-of-function toxicity. This model provides for simple and rapid screening of
novel ALS-linked PFN1 variants. To gain insight into potential novel roles for
profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with
yeast cells lacking profilin (pfy1Δ). Unexpectedly, deletion of several stress
granule and processing body genes, including pbp1Δ, were found to be synthetic
lethal with pfy1Δ. Mutations in ATXN2, the human ortholog of PBP1, are a known
ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian
cells. Given this genetic interaction and recent evidence linking stress granule
dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also
associate with stress granules. Here we report that profilin 1 and related
protein profilin 2 are novel stress granule-associated proteins in mouse primary
cortical neurons and in human cell lines and that ALS-linked mutations in
profilin 1 alter stress granule dynamics, providing further evidence for the
potential role of stress granules in ALS pathogenesis. Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders
including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes
for familial ALS, encodes a multifunctional DNA/RNA binding protein
predomitly localized in the nucleus. C-terminal mutations in FUS/TLS cause
the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the
present study, we examined the effects of ALS-linked FUS mutants on
ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants
were diffusely mislocalized in the cytoplasm as small granules in transiently
transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in
∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were
assembled into stress granules under stress conditions, and these were also
recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These
aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble
fraction, which also reduced the number of nuclear oligo(dT)-positive foci
(speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the
number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS
P525L. These findings raise the possibility that ALS-linked C-terminal FUS
mutants could sequester a variety of RNA binding proteins and mRNAs in the
cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium
and biogenesis. BACKGROUND: Mutations in calcium-responsive transactivator (CREST) encoding gene
have been recently linked to ALS. Similar to several proteins implicated in ALS,
CREST contains a prion-like domain and was reported to be a component of
paraspeckles.
RESULTS: We demonstrate that CREST is prone to aggregation and co-aggregates
with FUS but not with other two ALS-linked proteins, TDP-43 and TAF15, in
cultured cells. Aggregation of CREST affects paraspeckle integrity, probably by
trapping other paraspeckle proteins within aggregates. Like several other
ALS-associated proteins, CREST is recruited to induced stress granules. Neither
of the CREST mutations described in ALS alters its subcellular localization,
stress granule recruitment or detergent solubility; however Q388stop mutation
results in elevated steady-state levels and more frequent nuclear aggregation of
the protein. Both wild-type protein and its mutants negatively affect neurite
network complexity of unstimulated cultured neurons when overexpressed, with
Q388stop mutation being the most deleterious. When overexpressed in the fly eye,
wild-type CREST or its mutants lead to severe retinal degeneration without
obvious differences between the variants.
CONCLUSIONS: Our data indicate that CREST and certain other ALS-linked proteins
share several features implicated in ALS pathogenesis, namely the ability to
aggregate, be recruited to stress granules and alter paraspeckle integrity. A
change in CREST levels in neurons which might occur under pathological
conditions would have a profound negative effect on neuronal homeostasis. |
Mutations of which genes have been associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)? | Mutations in five genes – ryanodine receptor 2 (RYR2), calsequestrin 2(CASQ2), triadic (TRDN), calmodulin 1 (CALM1) and potassium channel, inwardly rectifying subfamily J, member 2 (KCNJ2) – have been found to be associated with CPVT | Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial
arrhythmogenic disorder associated with mutations in the cardiac ryanodine
receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro
studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular
Ca(2+)-signaling complex; however, direct evidence for such interactions and
their potential significance to myocardial function remain to be determined. We
identified a novel CASQ2 mutation in a young female with a structurally normal
heart and unexplained syncopal episodes. This mutation results in the
nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2
(R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led
to an increase in excitation-contraction coupling gain and to more frequent
occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals
(sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As
revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of
permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and
waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombit
CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro;
however, the mutant protein lacked the ability of its WT counterpart to inhibit
RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that
the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex
and impairs regulation of RyR2 by luminal Ca2+. These results show that
intracellular Ca2+ cycling in normal heart relies on an intricate interplay of
CASQ2 with the proteins of the RyR2 channel complex and that disruption of these
interactions can lead to cardiac arrhythmia. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic disease so far related to mutations in the cardiac ryanodine
receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in
RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations
in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT
patients with no detected mutation in these two genes. Based on a candidate gene
approach, we focused our investigations on triadin and junctin, two proteins
that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin
(ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three
mutations in triadin which cosegregated with the disease on a recessive mode of
transmission in two families, but no mutation was found in junctin. Two TRDN
mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop
codons; the third mutation, a p.T59R missense mutation, was further studied.
Expression of the p.T59R mutant in COS-7 cells resulted in intracellular
retention and degradation of the mutant protein. This was confirmed after in
vivo expression of the mutant triadin in triadin knock-out mice by viral
transduction. In this work, we identified TRDN as a new gene responsible for an
autosomal recessive form of CPVT. The mutations identified in the two families
lead to the absence of the protein, thereby demonstrating the importance of
triadin for the normal function of the cardiac calcium release complex in
humans. BACKGROUND: Mutations of KCNJ2, the gene encoding the human inward rectifier
potassium channel Kir2.1, cause Andersen-Tawil syndrome (ATS), a disease
exhibiting ventricular arrhythmia, periodic paralysis, and dysmorphic features.
However, some KCNJ2 mutation carriers lack the ATS triad and sometimes share the
phenotype of catecholaminergic polymorphic ventricular tachycardia (CPVT). We
investigated clinical and biophysical characteristics of KCNJ2 mutation carriers
with "atypical ATS."
METHODS AND RESULTS: Mutational analyses of KCNJ2 were performed in 57 unrelated
probands showing typical (≥2 ATS features) and atypical (only 1 of the ATS
features or CPVT) ATS. We identified 24 mutation carriers. Mutation-positive
rates were 75% (15/20) in typical ATS, 71% (5/7) in cardiac phenotype alone,
100% (2/2) in periodic paralysis, and 7% (2/28) in CPVT. We divided all carriers
(n=45, including family members) into 2 groups: typical ATS (A) (n=21, 47%) and
atypical phenotype (B) (n=24, 53%). Patients in (A) had a longer QUc interval
[(A): 695 ± 52 versus (B): 643 ± 35 ms] and higher U-wave amplitude (0.24 ± 0.07
versus 0.18 ± 0.08 mV). C-terminal mutations were more frequent in (A) (85%
versus 38%, P<0.05). There were no significant differences in incidences of
ventricular tachyarrhythmias. Functional analyses of 4 mutations found in (B)
revealed that R82Q, R82W, and G144D exerted strong domit negative suppression
(current reduction by 95%, 97%, and 96%, respectively, versus WT at -50 mV) and
T305S moderate suppression (reduction by 89%).
CONCLUSIONS: KCNJ2 gene screening in atypical ATS phenotypes is of clinical
importance because more than half of mutation carriers express atypical
phenotypes, despite their arrhythmia severity. BACKGROUND: The genetic background of catecholaminergic polymorphic ventricular
tachycardia (CPVT) has been extensively investigated for the last decade in
Western countries, but it remains unstudied in the Asian population.
METHODS AND RESULTS: In 50 Japanese probands from unrelated families who
satisfied clinical criteria for CPVT, genetic testing was conducted in all exons
on 3 CPVT-related genes: cardiac ryanodine receptor 2 (RYR2), calsequestrin 2
(CASQ2) and inward rectifier potassium channel 2 (KCNJ2), and the clinical
features between RYR2-genotyped and -non-genotyped patient groups were compared.
Genetic and clinical evaluation was also done in 46 family members. In the
genetic screening, 28 (18 novel) RYR2 (56.0%), 1 compound heterozygous CASQ2
(2.0%) and 1 KCNJ2 (2.0%) mutation carriers were identified. In the RYR2
mutation-positive group, the frequency of bidirectional ventricular tachycardia
and the use of β-blockers were significantly higher than in the
mutation-negative group. In contrast, there was no significant difference in
supraventricular arrhythmias between the 2 groups. With regard to disease
penetrance, the number of family members of RYR2-genotyped probands with a
clinical diagnosis of CPVT was high.
CONCLUSIONS: Thirty gene mutation carriers were found for 3 genes in 50 probands
clinically diagnosed as having CPVT. The penetrance of CPVT phenotype was
significantly higher in RYR2 mutation carriers, thus RYR2 gene screening in CPVT
patients would be indispensable to prevent unexpected cardiac sudden death of
young family members. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias
induced by physical or emotional stress, in the absence structural heart
abnormalities. The arrhythmias may cause syncope or degenerate into cardiac
arrest and sudden death which usually occurs during childhood. Recent studies
have shown that CPVT is caused by mutations in the cardiac ryanodine receptor
type 2 (RyR2) or calsequestrin 2 (CASQ2) genes. Both proteins are key
contributors to the intracellular Ca(2+) handling process and play a pivotal
role in Ca(2+) release from the sarcoplasmic reticulum to the cytosol during
systole. Although the molecular pathogenesis of CPVT is not entirely clear, it
was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum
Ca(2+) leak, which initiates delayed afterdepolarizations (DADs) and triggered
arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced
pluripotent stem cells (iPSC) generated from somatic cells (e.g. fibroblasts,
keratinocytes) now enables researches to investigate mutated cardiomyocytes
generated from the patient's iPSC. To this end, in the present article we review
recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the
mutated cells catecholamine-induced DADs and triggered arrhythmias. Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic
reticulum (SR) that provide the majority of calcium ions (Ca(2+)) necessary to
induce contraction of cardiac and skeletal muscle cells. In their intracellular
environment, RyR channels are regulated by a variety of cytosolic and luminal
factors so that their output signal (Ca(2+)) induces finely-graded cell
contraction without igniting cellular processes that may lead to aberrant
electrical activity (ventricular arrhythmias) or cellular remodeling. The
importance of RyR dysfunction has been recently highlighted with the
demonstration that point mutations in RYR2, the gene encoding for the cardiac
isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic
ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the
development of adrenergically-mediated ventricular tachycardia in individuals
with an apparently normal heart. Here we summarize the state of the field in
regards to the main arrhythmogenic mechanisms triggered by RyR2 channels
harboring mutations linked to CPVT. Most CPVT mutations characterized to date
endow RyR2 channels with a gain of function, resulting in hyperactive channels
that release Ca(2+) spontaneously, especially during diastole. The spontaneous
Ca(2+) release is extruded by the electrogenic Na(+)/Ca(2+) exchanger, which
depolarizes the external membrane (delayed afterdepolarization or DAD) and may
trigger untimely action potentials. However, a rare set of CPVT mutations yield
RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli,
and it is unclear whether these channels release Ca(2+) spontaneously during
diastole. We discuss novel cellular mechanisms that appear more suitable to
explain ventricular arrhythmias due to RyR2 loss-of-function mutations. |
What is the relationship between nucleosomes and exons? | Nucleosomes are preferentially located within exons compared to nearby intronic sequences. Preferential positioning within the exons is indepedent of gene expression levels, stronger in exons with weak splice sites and conserved through metazoan evolution. | We describe the structural implications of a periodic pattern found in human
exons and introns by hidden Markov models. We show that exons (besides the
reading frame) have a specific sequential structure in the form of a pattern
with triplet consensus non-T(A/T)G, and a minimal periodicity of roughly ten
nucleotides. The periodic pattern is also present in intron sequences, although
the strength per nucleotide is weaker. Using two independent profile methods
based on triplet bendability parameters from DNase I experiments and nucleosome
positioning data, we show that the pattern in multiple alignments of internal
exon and intron sequences corresponds to a periodic "in phase" bending potential
towards the major groove of the DNA. The nucleosome positioning data show that
the consensus triplets (and their complements) have a preference for locations
on a bent double helix where the major groove faces inward and is compressed.
The in-phase triplets are located adjacent to GCC/GGC triplets known to have the
strongest bias in their positioning on the nuclesome. Analysis of mRNA sequences
encoding proteins with known tertiary structure exclude the possibility that the
pattern is a consequence of the previously well-known periodicity caused by the
encoding of alpha-helices in proteins. Finally, we discuss the relation between
the bending potential of coding and non-coding regions and its impact on the
translational positioning of nucleosomes and the recognition of genes by the
transcriptional machinery. The characteristic AA(TT) sequence pattern of the nucleosome DNA derived earlier
is used for prediction of nucleosome positions around splice junctions of
eukaryotic genes. Two large datasets (2000 sequences each) were collected
consisting of DNA segments with the exon/intron and intron/exon splice
junctions, from various eukaryotic species. Positions of predicted nucleosomes
near the junction sites were calculated. Those junctions which are found to
belong to the nucleosomes, are located preferentially within a few base pairs
from the midpoint of the nucleosome DNA. That is, obligatory GT- and AG-ends of
the introns are more frequently located near the nucleosome dyad axis, within
the best protected middle 10-15 base pairs of the nucleosome DNA. In addition, a
tendency is observed for the strongest nucleosomes to form more often in the
introns, in accordance with the hypothesis on the chromatin-organizing role of
introns. A program for constructing nucleosome formation potential profile was applied
for investigation of exons, introns, and repetitive sequences. The program is
available at http://wwwmgs.bionet.nsc.ru/mgs/programs/recon/. We have
demonstrated that introns and repetitive sequences exhibit higher nucleosome
formation potentials than exons. This fact may be explained by functional
saturation of exons with genetic code, hindering the localization of efficient
nucleosome positioning sites. Gene sequences in the vicinity of splice sites are found to possess dinucleotide
periodicities, especially RR and YY, with the period close to the pitch of
nucleosome DNA. This confirms previously reported findings about preferential
positioning of splice junctions within the nucleosomes. The RR and YY
dinucleotides oscillate counter-phase, i.e., their respective preferred
positions are shifted about half-period from one another, as it was observed
earlier for AA and TT dinucleotides. Species specificity of nucleosome
positioning DNA pattern is indicated by the predomit use of the periodical
GG(CC) dinucleotides in human and mouse genes, as opposed to predomit AA(TT)
dinucleotides in Arabidopsis and C. elegans. Chromatin structure influences transcription, but its role in subsequent RNA
processing is unclear. Here we present analyses of high-throughput data that
imply a relationship between nucleosome positioning and exon definition. First,
we have found stable nucleosome occupancy within human and Caenorhabditis
elegans exons that is stronger in exons with weak splice sites. Conversely, we
have found that pseudoexons--intronic sequences that are not included in mRNAs
but are flanked by strong splice sites--show nucleosome depletion. Second, the
ratio between nucleosome occupancy within and upstream from the exons correlates
with exon-inclusion levels. Third, nucleosomes are positioned central to exons
rather than proximal to splice sites. These exonic nucleosomal patterns are also
observed in non-expressed genes, suggesting that nucleosome marking of exons
exists in the absence of transcription. Our analysis provides a framework that
contributes to the understanding of splicing on the basis of chromatin
architecture. The genomes of higher organisms are packaged in nucleosomes with functional
histone modifications. Until now, genome-wide nucleosome and histone
modification studies have focused on transcription start sites (TSSs) where
nucleosomes in RNA polymerase II (RNAPII) occupied genes are well positioned and
have histone modifications that are characteristic of expression status. Using
public data, we here show that there is a higher nucleosome-positioning signal
in internal human exons and that this positioning is independent of expression.
We observed a similarly strong nucleosome-positioning signal in internal exons
of Caenorhabditis elegans. Among the 38 histone modifications analyzed in man,
H3K36me3, H3K79me1, H2BK5me1, H3K27me1, H3K27me2, and H3K27me3 had evidently
higher signals in internal exons than in the following introns and were clearly
related to exon expression. These observations are suggestive of roles in
splicing. Thus, exons are not only characterized by their coding capacity, but
also by their nucleosome organization, which seems evolutionarily conserved
since it is present in both primates and nematodes. Nucleosome positioning is constrained at eukaryotic transcription start sites
and implicated in transcriptional regulation. Moreover, recent observations
indicate that chromatin structure, transcription and splicing are functionally
intertwined, and that modified nucleosomes with trimethylation of lysine 36 in
histone subunit 3 (H3K36me3) are enriched at internal exons and the downstream
flanking intronic regions of highly expressed genes. However, the position of
nucleosomes in the interior of genes has been thought to be largely random. Here
we show, by analysis of data sets from human sperm and T cells and medaka
(Japanese killifish, Oryzias latipes) blastulae, that internal exons of genes
are characterized by sharply elevated average nucleosome occupancy in comparison
to flanking intronic sequences. We also show that the preferential positioning
of nucleosomes at internal exons is independent of their modification status,
and of the GC content, conservation or the expression level of the exon. These
findings show that the location of exons is recorded in the chromatin structure
and may be inherited across generations. Such embedded information may underpin
transcriptionally coupled exon recognition and splice site selection. The occupancy of nucleosomes along chromosome is a key factor for gene
regulation. However, except promoter regions, genome-wide properties and
functions of nucleosome organization remain unclear in mammalian genomes. Using
the computational model of Increment of Diversity with Quadratic Discrimit
(IDQD) trained from the microarray data, the nucleosome occupancy score
(NOScore) was defined and applied to splice junction regions of constitutive,
cassette exon, alternative 3' and 5' splicing events in the human genome. We
found an interesting relation between NOScore and RNA splicing: exon regions
have higher NOScores compared with their flanking intron sequences in both
constitutive and alternative splicing events, indicating the stronger nucleosome
occupation potential of exon regions. In addition, NOScore valleys present at
approximately 25 bp upstream of the acceptor site in all splicing events. By
defining folding diversity-to-energy ratio to describe RNA structural
flexibility, we demonstrated that primary RNA transcripts from nucleosome
occupancy regions are relatively rigid and those from nucleosome depleted
regions are relatively flexible. The negative correlation between nucleosome
occupation/depletion of DNA sequence and structural flexibility/rigidity of its
primary transcript around splice junctions may provide clues to the deeper
understanding of the unexpected role for nucleosome organization in the
regulation of RNA splicing. Alternative splicing affects more than 90% of human genes. Coupling between
transcription and splicing has become crucial in the complex network underlying
alternative splicing regulation. Because chromatin is the real template for
nuclear transcription, changes in its structure, but also in the "reading" and
"writing" of the histone code, could modulate splicing choices. Here, we discuss
the evidence supporting these ideas, from the first proposal of chromatin
affecting alternative splicing, performed 20 years ago, to the latest findings
including genome-wide evidence that nucleosomes are preferentially positioned in
exons. We focus on two recent reports from our laboratories that add new
evidence to this field. The first report shows that a physiological stimulus
such as neuron depolarization promotes intragenic histone acetylation (H3K9ac)
and chromatin relaxation, causing the skipping of exon 18 of the neural cell
adhesion molecule gene. In the second report, we show how specific histone
modifications can be created at targeted gene regions as a way to affect
alternative splicing: Using small interfering RNAs (siRNAs), we increased the
levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the
human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA)
through a mechanism that recalls RNA-mediated transcriptional gene silencing. Recent studies indicate that precursor mRNA splicing occurs cotranscriptionally
and that nucleosome positioning may influence inclusion of internal exon
sequences. In eukaryotic genomes, most genes are subject to alternative
splicing. Nucleosome occupancy in sequences of different types of alternative
events was investigated by analyzing genome-wide nucleosome positioning data
sets from human. Nucleosomes were found to be preferentially positioned within
constitutive exons and/or constitutive portions of alternative exons, which was
not associated with gene expression or states of cells but was based on sequence
and positively related with the sequence conservation of splicing sites. In
addition, the nucleosome distribution in an alternative acceptor exon was
different from that in an alternative donor exon. Nucleosome occupancy around
constitutive transcription start sites also showed stronger +1 nucleosome
enrichment and weaker nucleosome occupancy in nucleosome free regions. These
results provided a new approach for better appreciation of the connection
between chromatin structure and gene expression regulation. If we define a genetic code as a widespread DNA sequence pattern that carries a
message with an impact on biology, then there are multiple genetic codes.
Sequences involved in these codes overlap and, thus, both interact with and
constrain each other, such as for the triplet code, the intron-splicing code,
the code for amphipathic alpha helices, and the chromatin code. Nucleosomes
preferentially are located at the ends of exons, thus protecting splice
junctions, with the N9 positions of guanines of the GT and AG junctions oriented
toward the histones. Analysis of protein-coding sequences reveals numerous
traces of tandem repeats, apparently formed by triplet expansion, which in
effect is a genome inflation ``code''. Our data are consistent with the
hypothesis that expansion of simple tandem repetition of certain aggressive
triplets has been a characteristic of life from its emergence. Such expanding
triplets appear to be the major factor underlying observed codon usage biases. |
List programs suitable for pharmacophore modelling | A pharmacophore is an abstract description of molecular features which are necessary for molecular recognition of a ligand by a biological macromolecule. The IUPAC defines a pharmacophore to be "an ensemble of steric and electronic features that is necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response". A pharmacophore model explains how structurally diverse ligands can bind to a common receptor site. Furthermore pharmacophore models can be used to identify through denovo design or virtual screening novel ligands that will bind to the same receptor. Nowadays there are many programs suitable for pharmacophore modelling such as LigandScout, Discovery Studio, Catalyst, PharmaGist, Genetic Algorithm Similarity and Molecular Operating Environment. | In the present study we investigate whether augmentation of pharmacophores with
excluded (ligand-inaccessible) volumes can condense the lengthy unspecific hit
lists often obtained in 3D-database searching. Our pharmacophores contained
hydrophobic features defined by the hormone, hydrogen bond donor and acceptor
features of the liganded rat THR-alpha X-ray structure, and excluded volumes
located at the positions and scaled according to the sizes of atoms delineating
the binding cavity. We now show, for the first time, that it is perfectly
feasible with the Catalyst software to search, in 1-2 h, medium-sized databases
such as Maybridge (with 5 x 10(5) compounds registered as multiple conformers)
with pharmacophores containing numerous (approximately 10(2)) excluded volumes.
The excluded volumes did not slow the search significantly; for pharmacophores
containing more features they also reduced the size of the hit list the most.
For example, with a 7-feature pharmacophore, the Maybridge hit list shrank from
4 to 1. The single remaining compound was subsequently shown to bind to
THR-alpha with an IC50 of 69 microM. Thus, we conclude that structure-based
pharmacophores augmented with numerous excluded volumes can effectively prune
and focus hit lists. The performance of multiple excluded volume-supplemented
structure-based pharmacophores in 3D-database mining as implemented with the
Catalyst software compares very favorably with other published procedures, with
respect to speed, specificity, and ease of use. The programs Phase and Catalyst HypoGen are compared for their performance in
determining three-dimensional quantitative structure-activity relationships.
Eight sets of compounds with measured activity were collected from the public
literature and partitioned into suitable training and test sets by an automated
procedure. A range of models is built with each program, and suggested parameter
variations are investigated. The models are assessed by their ability to predict
the activity of compounds in the test set, and it is demonstrated that the
performance of Phase is better than or equal to that of Catalyst HypoGen, with
the data sets and parameters used here. Additionally, compounds in two of the
data sets are overlaid on crystallographic structures of similar ligands in
complex with the target receptor, in order to guide pharmacophore generation by
the two programs, but the resulting models do not perform better. Computational conformational sampling is integral to small molecule
pharmaceutical research, for detailed conformational analysis and
high-throughput 3D library enumeration. These two regimes were tested in details
for the general-purpose modeling program MOE, using its three conformational
sampling methods, i.e. systematic search, stochastic search, and Conformation
Import. The tests include i) identification of the global energy minimum, ii)
reproduction of the bioactive conformation, iii) measures of conformational
coverage with 3D descriptors, and iv) compute times. The bioactive conformers
are from a new set of 256 diverse, druglike, protein-bound ligands compiled and
analyzed with particular care. The MOE results are compared to those obtained
from the established program Catalyst. Key parameters controlling the
conformational coverage were varied systematically. Coverage and diversity of
the conformational space were characterized with unique pharmacophore triplets
or quadruplets. Overall, the protocols in both MOE and Catalyst performed well
for their intended tasks. MOE performed at least as well as Catalyst for
high-throughput library generation and detailed conformational modeling. This
work provides a guide and specific recommendations regarding the usage of
conformational sampling tools in MOE. A 3D pharmacophore model had been generated for a series of dipeptide proteasome
inhibitors containing boron atoms using Catalyst. A data set consisting of 24
inhibitors was selected on the basis of the information content of the
structures and activity data as required by the Catalyst/HypoGen program. The
built model was able to predict the activity of other known proteasome
inhibitors not included in the model generation. Based on the analysis of the
best hypotheses, some novel proteasome inhibitors were designed and predicted.
Three dipeptide boronic acid inhibitors SMNT 1, SMNT 2, and SMNT 3, were
synthesized and biologically assayed. It turned out that the three designed
molecules were all more potent than the marketed MG341, and the experimental
values were consistent with the predicted ones, indicating that the theoretical
model was reliable enough to predict and design novel proteasome inhibitors. The
covalent interaction mode between the boron atom of the inhibitor and
O(gamma)-Thr1 residue of the 20S proteasome was studied for the first time by
employing the most potent inhibitor SMNT 2 with the Insight II 2005/Affinity
program. The docking results agreed well with the experimental ones. Three-dimensional pharmacophore models were generated for AT1 and ET(A)
receptors based on highly selective AT1 and ET(A) antagonists using the program
Catalyst/HipHop. Both the best pharmacophore model for selective AT1 antagonists
(Hypo-AT(1)-7) and ETA antagonists (Hypo-ET(A)-1) were obtained through a
careful validation process. All five features contained in Hypo-AT(1)-7 and
Hypo-ET(A)-1 (hydrogen-bond acceptor (A), hydrophobic aliphatic (Z), negative
ionizable (N), ring aromatic (R), and hydrophobic aromatic (Y)) seem to be
essential for antagonists in terms of binding activity. Dual AT1 and ET(A)
receptor antagonists (DARAs) can map to both Hypo-AT(1)-7 and Hypo-ET(A)-1,
separately. Comparison of Hypo-AT(1)-7 and Hypo-ET(A)-1, not only AT1 and ET(A)
antagonist pharmacophore models consist of essential features necessary for
compounds to be highly active and selective toward their corresponding receptor,
but also have something in common. The results in this study will act as a
valuable tool for designing and researching structural relationship of novel
dual AT1 and ET(A) receptor antagonists. Erectile dysfunction (ED) is a sexual disorder mainly caused by decrease in
cellular concentration of cyclic guanosine monophosphate (cGMP), which is
degraded by phosphodiesterase type-5 (PDE-5). As a potent therapeutic target,
inhibitors such as Viagra , Cialis, and Levitra have already been developed to
target PDE-5 for treating ED; traditional Chinese medicine, Epimedium
sagittatum, also has shown prominent results as well. To developed new PDE-5
inhibitors, we performed a virtual screening of traditional Chinese medicine
(TCM) database and docking analyses to identify candidates. Known PDE-5
inhibitors were used to construct a three dimensional quantitative
structure-activity relationship (3D QSAR) model by HypoGen program. From docking
analyses, isochlorogenic acid b was identified as the most potential inhibitory
compound. De novo evolution designed 47 derivatives. Of the 47 derivatives,
seven were able to map into the pharmacophore model, and these seven compounds
were suggested to be the most promising leads for inhibiting PDE-5. An analysis
of the hydrogen bond interactions formed between the docked ligands and PDE-5
identified ASN662, SER663 and GLN817 as the most frequently interacting
residues. A total of eight novel leading compounds were identified to have
favorable interaction with PDE-5. These compounds all had hydrogen bond
interactions with three key residues that could be further investigated for
understanding of PDE-5 and ligands interaction. Cholesteryl ester transfer protein (CETP) is involved in trafficking lipoprotein
particles and neutral lipids between HDL and LDL and therefore is considered a
valid target for treating dyslipidemic conditions and complications.
Pharmacophore modeling and quantitative structure-activity relationship (QSAR)
analysis were combined to explore the structural requirements for potent CETP
inhibitors. Two pharmacophores emerged in the optimal QSAR equation (r(2)=0.800,
n=96, F=72.1, r(2)(LOO) =0.775, r(2)(PRESS) against 22 external test
inhibitors=0.707) suggesting the existence of at least two distinct binding
modes accessible to ligands within CETP binding pocket. The successful
pharmacophores were complemented with strict shape constraints in an attempt to
optimize their receiver-operating characteristic (ROC) curve profiles. The
validity of our modeling approach was experimentally established by the
identification of several CETP inhibitory leads retrieved via in silico
screening of the National Cancer Institute (NCI) list of compounds and an in
house built database of drugs and agrochemicals. Two hits illustrated low
micromolar IC(50) values: NSC 40331 (IC(50)=6.5 microM) and NSC 89508
(IC(50)=1.9 microM). Active hits were then used to guide synthetic exploration
of a new series of CETP inhibitors. To probe the selective mechanism of agonists binding to three opioid receptor
subtypes, ligand-based and receptor-based methods were implemented together and
subtype characteristics of opioid agonists were clearly described. Three
pharmacophore models of opioid agonists were generated by the Catalyst/HypoGen
program. The best pharmacophore models for μ, δ and κ agonists contained four,
five and five features, respectively. Meanwhile, the three-dimensional
structures of three receptor subtypes were modeled on the basis of the crystal
structure of β2-adrenergic receptor, and molecular docking was conducted
further. According to these pharmacophore models and docking results, the
similarities and differences among agonists of three subtypes were identified. μ
or δ agonists, for example, could form one hydrogen bond separately with Tyr129
and Tyr150 at TMIII, whereas κ ones formed a π-π interaction in that place.
These findings may be crucial for the development of novel selective analgesic
drugs. The pharmacophore model (Hypo1) with a well prediction capacity for CysLT(1)
antagonists was developed using Catalyst/HypoGen program. Virtual screening
against an in-house database consisted of carboxylated chalcones using Hypo1 was
performed. Retrieved hits 26a, 26b, 27a, and 27b were synthesized and biological
evaluated, the results of which demonstrated that these compounds showed
moderate to good CysLT(1) antagonistic activities. This study indicated that the
generated model (Hypo1) is a reliable and useful tool in lead optimization for
novel CysLT(1) antagonists. In an attempt to identify potential HCV NS3 protease inhibitors lead compounds,
a series of novel indoles (10a-g) was designed. Molecular modeling study,
including fitting to a 3D-pharmacophore model of the designed molecules (10a-g),
with HCV NS3 protease hypothesis using catalyst program was fulfilled. Also, the
molecular docking into the NS3 active site was examined using Discovery Studio
2.5 software. Several compounds showed significant high simulation docking score
and fit values. The designed compounds with high docking score and fit values
were synthesized and biologically evaluated in vitro using an NS3 protease
binding assay. It appears that most of the tested compounds reveal promising
inhibitory activity against NS3 protease. Of these, compounds 10a and 10b
demonstrated potent HCV NS3 protease inhibitors with IC(50) values of 9 and
12μg/mL, respectively. The experimental serine protease inhibitor activities of
compounds 10a-g were consistent with their molecular modeling results.
Inhibitors from this class have promising characteristics for further
development as anti-HCV agents. Pancreatic cholesterol esterase (CEase) is a serine hydrolase involved in the
hydrolysis of variety of lipids and transport of free cholesterol. In this
study, pharmacophore hypotheses based on known inhibitors were generated using
common feature pharmacophore generation protocol available in Discovery Studio
program. The best pharmacophore model containing two hydrogen bond acceptor and
three hydrophobic features was selected and validated. It was further used in
screening three diverse chemical databases. Hit compounds were subjected to
drug-likeness and molecular docking studies. Four hits, namely SEW00846,
NCI0040784, GK03167, and CD10645, were selected based on the GOLD fitness score
and interaction with active site amino acids. All hit compounds were further
optimized to improve their binding in the active site. The optimized compounds
were found to have improved binding at the active site. Strongly binding
optimized hits at the active site can act as virtual leads in potent CEase
inhibitor designing. A set of 5-phenyl-1-(3-pyridyl)-1H-1,2,4-triazole-3-carboxylic acid derivatives
(16â32) showing anti-inflammatory activity was analyzed using a
three-dimensional qualitative structure-selectivity relationship (3D QSSR)
method. The CatalystHipHop approach was used to generate a pharmacophore model
for cyclooxygenase-2 (COX-2) inhibitors based on a training set of 15 active
inhibitors (1â15). The degree of fitting of the test set compounds (16â32) to
the generated hypothetical model revealed a qualitative measure of the more or
less selective COX-2 inhibition of these compounds. The results indicate that
most derivatives (16, 18, 20â25, and 30â32) are able to effectively satisfy the
proposed pharmacophore geometry using energy accessible conformers (E(conf) < 20
kcal/mol). In addition, the triazole derivatives (16â32) were docked into COX-1
and COX-2 X-ray structures, using the program GOLD. Based on the docking results
it is suggested that several of these novel triazole derivatives are active COX
inhibitors with a significant preference for COX-2. In principle, this work
presents an interesting, comprehensive approach to theoretically predict the
mode of action of compounds that showed anti-inflammatory activity in an in vivo
model. CCR3, a G protein-coupled receptor, plays a central role in allergic
inflammation and is an important drug target for inflammatory diseases. To
understand the structure-function relationship of CCR3 receptor, different
computational techniques were employed, which mainly include: (i) homology
modeling of CCR3 receptor, (ii) 3D-quantitative pharmacophore model of CCR3
antagonists, (iii) virtual screening of small compound databases, and (iv)
finally, molecular docking at the binding site of the CCR3 receptor homology
model. Pharmacophore model was developed for the first time, on a training data
set of 22 CCR3 antagonists, using CATALYST HypoRefine program. Best hypothesis
(Hypo1) has three different chemical features: two hydrogen-bond acceptors, one
hydrophobic, and one ring aromatic. Hypo1 model was further validated using (i)
87 test set CCR3 antagonists, (ii) Cat Scramble randomization technique, and
(iii) Decoy data set. Molecular docking studies were performed on modeled CCR3
receptor using 303 virtually screened hits, obtained from small compound
database virtual screening. Finally, five hits were identified as potential
leads against CCR3 receptor, which exhibited good estimated activities,
favorable binding interactions, and high docking scores. These studies provided
useful information on the structurally vital residues of CCR3 receptor involved
in the antagonist binding, and their unexplored potential for the future
development of potent CCR3 receptor antagonists. The proton-coupled amino acid transporter hPAT1 has recently gained much
interest due to its ability to transport small drugs thereby allowing their oral
administration. A three-dimensional quantitative structure-activity relationship
(3D QSAR) study has been performed on its natural and synthetic substrates
employing comparative molecular similarity indices analysis (CoMSIA) to
investigate the structural requirements for substrates and to derive a
predictive model that may be used for the design of new prodrugs. The
cross-validated CoMSIA models have been derived from a training set of 40
compounds and the predictive ability of the resulting models has been evaluated
against a test set of 10 compounds. Despite the relatively narrow range of
binding affinities (K(i) values) reliable statistical models with good
predictive power have been obtained. The best CoMSIA model in terms of a proper
balance of all statistical terms and the overall contribution of individual
properties has been obtained by considering steric, hydrophobic, hydrogen bond
donor and acceptor descriptors (q(cv)(2)=0.683, r(2)=0.958 and
r(PRED)(2)=0.666). The 3D QSAR model provides insight in the interactions
between substrates and hPAT1 on the molecular level and allows the prediction of
affinity constants of new compounds. A pharmacophore model has been generated
from the training set by means of the MOE (molecular operating environment)
program. This model has been used as a query for virtual screening to retrieve
potential new substrates from the small-molecule, 'lead-like' databases of MOE.
The affinities of the compounds were predicted and 11 compounds were identified
as possible high-affinity substrates. Two selected compounds strongly inhibited
the hPAT mediated l-[(3)H]proline uptake into Caco-2 cells constitutively
expressing the transport protein. Molecular docking is the most commonly used technique in the modern drug
discovery process where computational approaches involving docking algorithms
are used to dock small molecules into macromolecular target structures. Over the
recent years several evaluation studies have been reported by independent
scientists comparing the performance of the docking programs by using default
'black box' protocols supplied by the software companies. Such studies have to
be considered carefully as the docking programs can be tweaked towards optimum
performance by selecting the parameters suitable for the target of interest. In
this study we address the problem of selecting an appropriate docking and
scoring function combination (88 docking algorithm-scoring functions) for
substrate specificity predictions for feruloyl esterases, an industrially
relevant enzyme family. We also propose the 'Key Interaction Score System'
(KISS), a more biochemically meaningful measure for evaluation of docking
programs based on pose prediction accuracy. Sodium hydrogen exchanger (SHE) inhibitor is one of the most important targets
in treatment of myocardial ischemia. In the course of our research into new
types of non-acylguanidine, SHE inhibitory activities of
5-tetrahydroquinolinylidine aminoguanidine derivatives were used to build
pharmacophore and 3D-QSAR models. Genetic Algorithm Similarity Program (GASP)
was used to derive a 3D pharmacophore model which was used in effective
alignment of data set. Eight molecules were selected on the basis of structure
diversity to build 10 different pharmacophore models. Model 1 was considered as
the best model as it has highest fitness score compared to other nine models.
The obtained model contained two acceptor sites, two donor atoms and one
hydrophobic region. Pharmacophore modeling was followed by substructure
searching and virtual screening. The best CoMFA model, representing steric and
electrostatic fields, obtained for 30 training set molecules was statistically
significant with cross-validated coefficient (q(2)) of 0.673 and conventional
coefficient (r(2)) of 0.988. In addition to steric and electrostatic fields
observed in CoMFA, CoMSIA also represents hydrophobic, hydrogen bond donor and
hydrogen bond acceptor fields. CoMSIA model was also significant with
cross-validated coefficient (q(2)) and conventional coefficient (r(2)) of 0.636
and 0.986, respectively. Both models were validated by an external test set of
eight compounds and gave satisfactory prediction (r(pred)(2)) of 0.772 and 0.701
for CoMFA and CoMSIA models, respectively. This pharmacophore based 3D-QSAR
approach provides significant insights that can be used to design novel, potent
and selective SHE inhibitors. In this study, chemical feature based pharmacophore models of MMP-1, MMP-8 and
MMP-13 inhibitors have been developed with the aid of HypoGen module within
Catalyst program package. In MMP-1 and MMP-13, all the compounds in the training
set mapped HBA and RA, while in MMP-8, the training set mapped HBA and HY. These
features revealed responsibility for the high molecular bioactivity, and this is
further used as a three dimensional query to screen the knowledge based designed
molecules. These pharmacophore models for collagenases picked up some potent and
novel inhibitors. Subsequently, docking studies were performed for the potent
molecules and novel hits were suggested for further studies based on the docking
score and active site interactions in MMP-1, MMP-8 and MMP-13. In the present paper 25 known thermolysin inhibitors were docked into
thermolysin using the Internal Coordinate Mechanics (ICM) software.
Pharmacophore models based on thermolysin binding modes and activity profiles
were generated using the LigandScout program. The docking studies indicated that
all 25 inhibitors coordinated the catalytic zinc in bidentate or monodentate
geometry. A 'three-point' pharmacophore model was proposed which consisted of a
hydrophobic group, a negative ionizable group and a hydrogen bond acceptor
group. Finally the pharmacophore model has been tested against a small compound
library containing 18 highly, moderately, less active as well as inactive
compounds. The screening indicated that the pharmacophore model could, identify
highly active compounds in front of inactive or less active ones. Three-dimensional pharmacophore hypothesis was established based on a set of
known DPP-IV inhibitor using PharmaGist software program understanding the
essential structural features for DPP-IV inhibitor. The various marketed or
under developmental status, potential gliptins have been opted to build a
pharmacophore model, e.g. Sitagliptin (MK- 0431), Saxagliptin, Melogliptin,
Linagliptin (BI-1356), Dutogliptin, Carmegliptin, Alogliptin and Vildagliptin
(LAF237). PharmaGist web based program is employed for pharmacophore
development. Four points pharmacophore with the hydrogen bond acceptor (A),
hydrophobic group (H), Spatial Features and aromatic rings (R) have been
considered to develop pharmacophoric features by PharmaGist program. The best
pharmacophore model bearing the Score 16.971, has been opted to screen on
ZincPharmer database to derive the novel potential anti-diabetic ligands. The
best pharmacophore bear various Pharmacophore features, including General
Features 3, Spatial Features 1, Aromatic 1 and Acceptors 2. The PharmaGist
employed algorithm to identify the best pharmacophores by computing multiple
flexible alignments between the input ligands. The multiple alignments are
generated by combining alignments pair-wise between one of the gliptin input
ligands, which acts as pivot and the other gliptin as ligand. The resulting
multiple alignments reveal spatial arrangements of consensus features shared by
different subsets of input ligands. The best pharmacophore model has been
derived using both pair-wise and multiple alignment methods, which have been
weighted in Pharmacophore Generation process. The highest-scoring pharmacophore
model has been selected as potential pharmacophore model. In conclusion, 3D
structure search has been performed on the "ZincPharmer Database" to identify
potential compounds that have been matched with the proposed pharmacophoric
features. The 3D ZincPharmer Database has been matched with various thousands of
Ligands hits. Those matches were screened through the RMSD and max hits per
molecule. The physicochemical properties of various "ZincPharmer Database"
screened ligands have been calculated by PaDELDescriptor software. The all
"ZincPharmer Database" screened ligands have been filtered based on the
Lipinski's rule-of-five criteria (i.e. Molecular Weight < 500, H-bond acceptor ≤
10, H-bond donor ≤ 5, Log P ≤ 5) and were subjected to molecular docking studies
to get the potential antidiabetic ligands. We have found various substituted as
potential antidiabetic ligands, which can be used for further development of
antidiabetic agents. In the present research work, we have covered rational of
DPP-IV inhibitor based on Ligand-Based Pharmacophore detection, which is
validated via the Docking interaction studies as well as Maximal Common
Substructure (MCS). Antibiotic treatment may fail to protect individuals, if not started early
enough, after infection with Bacillus anthracis, due to the continuing activity
of toxins that the bacterium produces. Stable and easily stored inhibitors of
the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event
of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic
activity is to convert ATP to cAMP, and it is thus in principle a simple
phosphatase, which means that many mammalian enzymes, including intracellular
adenylcyclases, may have a similar activity. While nucleotide based inhibitors,
similar to its natural substrate, ATP, were identified early, these compounds
had low activity and specificity for EF. We used a combined structural and
computational approach to choose small organic molecules in large, web-based
compound libraries that would, based on docking scores, bind to residues within
the substrate binding pocket of EF. A family of fluorenone-based inhibitors was
identified that inhibited the release of cAMP from cells treated with EF. The
lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic
E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These
inhibitors are now being tested for their ability to inhibit Anthrax infection
in animal models and may have use against other pathogens that produce toxins
similar to EF, such as Bordetella pertussis or Vibrio cholera. |
Is selenium deficiency involved in autoimmune thyroid disease? | Selenium deficiency is likely to constitute a risk factor for a feedforward derangement of the immune system-thyroid interaction, while selenium supplementation appears to dampen the self-amplifying nature of this derailed interactionIn areas with severe selenium deficiency higher incidence of thyroiditis has been reported due to a decreased activity of selenium-dependent glutathione peroxidase enzyme within thyroid cells | In areas with severe selenium deficiency there is a higher incidence of
thyroiditis due to a decreased activity of selenium-dependent glutathione
peroxidase activity within thyroid cells. Selenium-dependent enzymes also have
several modifying effects on the immune system. Therefore, even mild selenium
deficiency may contribute to the development and maintece of autoimmune
thyroid diseases. We performed a blinded, placebo-controlled, prospective study
in female patients (n = 70; mean age, 47.5 +/- 0.7 yr) with autoimmune
thyroiditis and thyroid peroxidase antibodies (TPOAb) and/or Tg antibodies
(TgAb) above 350 IU/ml. The primary end point of the study was the change in
TPOAb concentrations. Secondary end points were changes in TgAb, TSH, and free
thyroid hormone levels as well as ultrasound pattern of the thyroid and quality
of life estimation. Patients were randomized into 2 age- and antibody
(TPOAb)-matched groups; 36 patients received 200 microg (2.53 micromol) sodium
selenite/d, orally, for 3 months, and 34 patients received placebo. All patients
were substituted with L-T(4) to maintain TSH within the normal range. TPOAb,
TgAb, TSH, and free thyroid hormones were determined by commercial assays. The
echogenicity of the thyroid was monitored with high resolution ultrasound. The
mean TPOAb concentration decreased significantly to 63.6% (P = 0.013) in the
selenium group vs. 88% (P = 0.95) in the placebo group. A subgroup analysis of
those patients with TPOAb greater than 1200 IU/ml revealed a mean 40% reduction
in the selenium-treated patients compared with a 10% increase in TPOAb in the
placebo group. TgAb concentrations were lower in the placebo group at the
beginning of the study and significantly further decreased (P = 0.018), but were
unchanged in the selenium group. Nine patients in the selenium-treated group had
completely normalized antibody concentrations, in contrast to two patients in
the placebo group (by chi(2) test, P = 0.01). Ultrasound of the thyroid showed
normalized echogenicity in these patients. The mean TSH, free T(4), and free
T(3) levels were unchanged in both groups. We conclude that selenium
substitution may improve the inflammatory activity in patients with autoimmune
thyroiditis, especially in those with high activity. Whether this effect is
specific for autoimmune thyroiditis or may also be effective in other endocrine
autoimmune diseases has yet to be investigated. Genetic factors play an important role in the pathogenesis of autoimmune thyroid
disease (AITD) and it has been calculated that 80% of the susceptibility to
develop Graves' disease is attributable to genes. The concordance rate for AITD
among monozygotic twins is, however, well below 1 and environmental factors thus
must play an important role. We have attempted to carry out a comprehensive
review of all the environmental and hormonal risk factors thought to bring about
AITD in genetically predisposed individuals. Low birth weight, iodine excess and
deficiency, selenium deficiency, parity, oral contraceptive use, reproductive
span, fetal microchimerism, stress, seasonal variation, allergy, smoking,
radiation damage to the thyroid gland, viral and bacterial infections all play a
role in the development of autoimmune thyroid disorders. The use of certain
drugs (lithium, interferon-alpha, Campath-1H) also increases the risk of the
development of autoimmunity against the thyroid gland. Further research is
warranted into the importance of fetal microchimerism and of viral infections
capable of mounting an endogenous interferon-alpha response. The essential trace mineral selenium is of fundamental importance to human
health. It is incorporated in the proteome in the forms of the genetically
encoded amino acids selenocysteine and selenomethionine, which are the
characteristic components of selenoproteins (SeP) such as glutathione
peroxidases (GPx), thioredoxin reductases and iodothyronine deiodinase families.
Thyroid is especially sensitive to selenium deficiency, because SeP can modify
thyreocytes function by acting as antioxidants and modifying redox status and
thyroid hormone metabolism. SeP are also involved in apoptosis, cell growth and
modification of the action of cell signalling systems and transcription factors.
Some intestinal GPx modulate apoptosis by removing the cells affected by
oxidative damage preserving tissue integrity. The malfunctioning of the GPx
antioxidant system in intestinal mucosa can trigger a continuous cycle of
reactive oxygen species and inflammation. Selenium deficiency is a risk factor,
due to the malabsorption, in celiac disease (CD) because the inflammatory damage
affects the small intestine; this deficiency can modulate SeP genes expression,
with consequent reiteration of inflammation and increase of mucosal damage. In
active CD, overexpression of interleukin-15 (IL-15) may increase activation of
effector mechanisms of epithelial damage by stimulating T helper 1 cytokine
proliferation and production and intraepithelial lymphocytes cytotoxicity by
protecting these lymphocytes from apoptosis. Blocking IL-15 has the potential to
provide new therapeutic tools to prevent both tissue damage and complication of
CD such as autoimmune thyroid diseases (AITD) where IL-15 expression is also
increases. In view of the role played by SeP in apoptosis inhibition, the
presence of environmental factors such as selenium deficiency can be considered
an important direct factor of thyroidal damage in development of AITD. Selenoproteins contain the essential trace element selenium whose deficiency
leads to major disorders including cancer, male reproductive system failure, or
autoimmune thyroid disease. Up to now, 25 selenoprotein-encoding genes were
identified in mammals, but the spatiotemporal distribution, regulation, and
function of some of these selenium-containing proteins remain poorly documented.
Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein, is
regulated by the trophic neuropeptide pituitary adenylate cyclase-activating
polypeptide (PACAP) in differentiating but not mature adrenomedullary cells. In
fact, our analysis revealed that, in rat, SelT is highly expressed in most
embryonic structures, and then its levels decreased progressively as these
organs develop, to vanish in most adult tissues. In the brain, SelT was
abundantly expressed in neural progenitors in various regions such as the cortex
and cerebellum but was undetectable in adult nervous cells except rostral
migratory-stream astrocytes and Bergmann cells. In contrast, SelT expression was
maintained in several adult endocrine tissues such as pituitary, thyroid, or
testis. In the pituitary gland, SelT was found in secretory cells of the
anterior lobe, whereas in the testis, the selenoprotein was present only in
spermatogenic and Leydig cells. Finally, we found that SelT expression is
strongly stimulated in liver cells during the regenerative process that occurs
after partial hepatectomy. Taken together, these data show that SelT induction
is associated with ontogenesis, tissue maturation, and regenerative mechanisms,
indicating that this PACAP-regulated selenoprotein may play a crucial role in
cell growth and activity in nervous, endocrine, and metabolic tissues. High prevalence of hyperplastic and autoimmune diseases of thyroid in Ukrainian
population is determined by endemic deficit of iodine and selenium. The aim of
this research was to assess the place of biologically-active additions on the
basis of herbal material containing an iodine and selenium in prophylaxis and
treatment of thyroid pathology. During the six month period 55 patients received
herbal preparation Alba twice a day. The levels of TSH, volume of thyroid, the
sizes of nodular goiter (ultrasound investigation) were measured before and at
the end of the investigation. The levels of thyroid stimulating antibodies to
TSH receptor (AB-r TSH) were evaluated in patients with hyperthyroidism. The
results of Alba application showed that in patients with thyroid pathology
(diffuse nontoxic goiter, hyperthyroidism and chronic thyroiditis) it was
possible to reduce the volume of thyroid, normalize its function, and decrease
the level of AB-r TSH in diffuse toxic goiter. We also found approximately 20 %
shortening of the time needed to get target level of TSH and finally the
duration of treatment of thyrotoxicosis. |
List disorders that are caused by mutations in the mitochondrial MTND6 gene. | Mitochondrial MTND6 gene mutations are the cause of Leigh syndrome and Leber's hereditary optic neuropathy and/or dystonia. | A novel point mutation in the ND6 subunit of complex I at position 14,459 of the
mitochondrial DNA (MTND6*LDY T14459A) was identified as a candidate mutation for
the highly tissue-specific disease. Leber's hereditary optic neuropathy plus
dystonia. Since the MTND6*LDYT14459A mutation was identified in a single family,
other pedigrees with the mutation are needed to confirm its association with the
disease. Clinical, biochemical, and genetic characterization is reported in two
additional pedigrees. Leber's hereditary optic neuropathy developed in two
family members in one pedigree. The daughter had clinically silent basal ganglia
lesions. In a second pedigree, a single individual presented with
childhood-onset generalized dystonia and bilateral basal ganglia lesions.
Patient groups that included individuals with Leigh's disease, dystonia plus
complex neurodegeneration, and Leber's hereditary optic neuropathy did not
harbor the MTND6*LDYT14459A mutation, suggesting that this mutation displays a
high degree of tissue specificity, thus producing a narrow phenotypic range.
These results confirm the association of the MTND6*LDYT14459A mutation with
Leber's hereditary optic neuropathy and/or dystonia. As the first genetic
abnormality that has been identified to cause generalized dystonia, this
mutation suggests that nuclear DNA or mitochondrial DNA mutations in oxidative
phosphorylation genes are important considerations in the pathogenesis of
dystonia. The objective of this study was to investigate clinical, biochemical, and
genetic features in 7 probands (a total of 11 patients) with nicotine-amide
adenine dinucleotide (NADH) dehydrogenase (complex I) deficiency. We screened
the mitochondrial DNA for mutations and found pathogenic mutations in complex I
genes (mitochondrial NADH dehydrogenase subunit (MTND) genes) in three probands.
The 10191T>C mutation in MTND3 and the 14487T>C mutation in MTND6 were present
in two probands with Leigh's-like and Leigh's syndrome, respectively. Four
siblings with a syndrome consisting of encephalomyopathy with hearing
impairment, optic nerve atrophy, and cardiac involvement had the 11778G>A
mutation in MTND4, previously associated with Leber hereditary optic neuropathy.
These findings demonstrate that mutations in MTND genes are relatively frequent
in patients with complex I deficiency. Biochemical measurements of respiratory
chain function in muscle mitochondria showed that all patients had a moderate
decrease of the mitochondrial adenosine triphosphate production rate.
Interestingly, the complex I deficiency caused secondary metabolic alterations
with decreased oxaloacetate-induced inhibition of succinate dehydrogenase
(complex II) and excretion of Krebs cycle intermediates in the urine. Our
results thus suggest that altered regulation of metabolism may play an important
role in the pathogenesis of complex I deficiency. PURPOSE: Leber hereditary optic neuropathy (LHON) is regarded as the most common
mitochondrial disease. We have previously reported comprehensive
population-based epidemiological data on common mitochondrial DNA (mtDNA)
mutations including m.3243A>G, m.8344A>G and large-scale mtDNA deletions in
Northern Finland. Our aim was to investigate the prevalence of primary LHON
mutations and mutations in the four mtDNA genes considered hot spots for LHON in
the same population.
METHODS: The study population consisted of 42 adult patients with an
aetiologically undefined bilateral optic atrophy. The major LHON mutations
m.3460G>A, m.11778G>A and m.14484T>C were analysed by restriction fragment
length polymorphism (RFLP), and MTND1, MTND6 and MTATP6 genes were sequenced.
MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE).
RESULTS: No major LHON mutations were found in the population of the province of
Northern Ostrobothnia giving the prevalence of these mutations 0-1.36:100 000
(95% CI). However, two main mutations were found elsewhere in Northern Finland,
homoplasmic m.11778G>A from Kainuu and heteroplasmic m.3460G>A from Central
Ostrobothnia. Furthermore, tobacco-alcohol amblyopia was diagnosed in five
patients in the study population and one of them had m.11778G>A.
CONCLUSION: The prevalence of the three major LHON mutations is lower in
Northern Finland than elsewhere in Finland or in Western Europe. As LHON and
tobacco-alcohol amblyopia have a similar phenotype, we recommend analysing the
known LHON-associated mutations before setting tobacco-alcohol amblyopia
diagnosis. IMPORTANCE: Mitochondrial DNA (mtDNA) disorders have emerged as major causes of
inherited neurologic disease. Despite being well recognized for more than 2
decades, the clinical presentation continues to broaden. The phenotypic
heterogeneity is partly owing to different percentage levels of mutant mtDNA
heteroplasmy in different tissues, but the factors influencing this are poorly
understood.
OBSERVATIONS: This case report describes monozygotic male twins with ptosis,
optic atrophy, and recent-onset intractable myoclonic epilepsy. The assessment
of respiratory chain enzyme activities in the muscle from 1 twin revealed a
severe and isolated defect involving mitochondrial complex I. Mitochondrial DNA
sequencing revealed a pathogenic m.14487T>C MTND6 mutation, which was present at
very high levels of heteroplasmy in muscle (84%) and lower levels in blood
(15%), urinary epithelium (75%), and buccal mucosa (58%). Of particular
interest, his identical twin was found to harbor very similar levels of the
m.14487T>C mutation in his blood, urine, buccal mucosa, and hair follicle DNA
samples, while the presence of low levels in the mother's tissues confirmed
maternal transmission.
CONCLUSIONS AND RELEVANCE: It was shown that m14487T>C can also cause the
unusual combination of optic atrophy, ptosis, and encephalomyopathy leading to
intractable seizures. Near-identical heteroplasmy levels in different tissues in
both siblings support a nuclear genetic mechanism controlling the tissue
segregation of mtDNA mutations. |
Which are the most common methods for ctDNA (circulating tumour DNA) detection? | Recently, nanoplasmonics has emerged as a platform for one-step dual detection with high sensitivity and specificity. The practice of "liquid biopsy" as a diagnostic, prognostic and theranostic tool in non-small cell lung cancer (NSCLC) patients is an appealing approach, at least in theory, since it is noninvasive and easily repeated. Cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. A new DNA sensor using a nickel(II) phenanthroline complex ([Ni(phen)(2)PHPIP]·2ClO(4)) as the electrochemical probe was developed. The sensor is very sensitive and selective for calf thymus DNA (ctDNA) detection in aqueous medium. | Author information:
(1)1] Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University, Stanford, California, USA. [2] Division of Oncology, Department of
Medicine, Stanford Cancer Institute, Stanford University, Stanford, California,
USA. [3].
(2)1] Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University, Stanford, California, USA. [2] Department of Radiation Oncology,
Stanford University, Stanford, California, USA. [3].
(3)Department of Radiation Oncology, Stanford University, Stanford, California,
USA.
(4)1] Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University, Stanford, California, USA. [2] Division of Oncology, Department of
Medicine, Stanford Cancer Institute, Stanford University, Stanford, California,
USA.
(5)Division of Oncology, Department of Medicine, Stanford Cancer Institute,
Stanford University, Stanford, California, USA.
(6)Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford
School of Medicine, Stanford University, Stanford, California, USA.
(7)1] Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University, Stanford, California, USA. [2] Division of Oncology, Department of
Medicine, Stanford Cancer Institute, Stanford University, Stanford, California,
USA. [3] Division of Hematology, Department of Medicine, Stanford Cancer
Institute, Stanford University, Stanford, California, USA.
(8)1] Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University, Stanford, California, USA. [2] Department of Radiation Oncology,
Stanford University, Stanford, California, USA. [3] Stanford Cancer Institute,
Stanford University, Stanford, California, USA. Circulating tumor DNA (ctDNA) bearing tumor-specific mutation and methylation
are promising biomarkers for noninvasive cancer assessment. However, existing
methods for ctDNA detection are restricted to genetic mutations. Recently,
oplasmonics has emerged as a platform for one-step dual detection with high
sensitivity and specificity. Here we present a strategy for ultrasensitive
detection of tumor-specific mutations (E542K and E545K) and methylation of ctDNA
of PIK3CA gene based on localized surface plasmon resoce (LSPR) and the
coupling plasmon mode of gold oparticles (AuNPs). Peptide nucleic acids (PNA)
is used as a probe to capture and enrich the 69-bp PIK3CA ctDNA. The exposure of
PNA-probed AuNPs to 200 fM ctDNA generates LSPR-peak shift of 4.3 nm,
corresponding to the primary response. Immunogold colloids are exploited as
methylation detectors and plasmon coupling based enhancement for secondary
response. LSPR-peak shifted from 4.3 nm to 11.4 nm upon the immunogold colloids
binding to two methylcytosines (mCpG), which is an approximately 107% increase,
compared to that of the primary response. This enhancement leads to four times
(~50 fM) improvement of sensitivity and because of two mCpG sites, ctDNA was
detected. These results demonstrate that the sensor can simultaneously detect
the hot-spot mutation and epigenetic changes on the ctDNA. Promisingly, other
specific-tumor mutants and epigenetic changes can be detected at low
concentration with this platform. The practice of "liquid biopsy" as a diagnostic, prognostic and theranostic tool
in non-small cell lung cancer (NSCLC) patients is an appealing approach, at
least in theory, since it is noninvasive and easily repeated. In particular,
this approach allows patient monitoring during treatment, as well as the
detection of different genomic alterations that are potentially accessible to
targeted therapy or are associated with treatment resistance. However, clinical
routine practice is slow to adopt the liquid biopsy. Several reasons may explain
this: (I) the vast number of methods described for potential detection of
circulating biomarkers, without a consensus on the ideal technical approach;
(II) the multiplicity of potential biomarkers for evaluation, in particular,
circulating tumor cells (CTCs) vs. circulating tumor DNA (ctDNA); (III) the
difficulty in controlling the pre-analytical phase to obtain robust and
reproducible results; (IV) the present cost of the currently available
techniques, which limits accessibility to patients; (V) the turnaround time
required to obtain results that are incompatible with the urgent need for
delivery of treatment. The purpose of this review is to describe the main
advances in the field of CTC and ctDNA detection in NSCLC patients and to
compare the main advantages and disadvantages of these two approaches. Circulating tumor DNA (ctDNA) is now being extensively studied as it is a
noninvasive "real-time" biomarker that can provide diagnostic and prognostic
information before, during treatment and at progression. These include DNA
mutations, epigenetic alterations and other forms of tumor-specific
abnormalities such as microsatellite instability (MSI) and loss of
heterozygosity (LOH). ctDNA is of great value in the process of cancer
treatment. However, up to date, there is no strict standard considering the
exact biomarker because the development and progression of cancer is extremely
complicated. Also, results of the studies evaluating ctDNA are not consistent
due to the different detection methods and processing. The major challenge is
still assay sensitivity and specificity for analysis of ctDNA. This review
mainly focuses on the tumor specific DNA mutations, epigenetic alterations as
well as detecting methods of ctDNA. The advantages and disadvantages will also
be discussed. |
Which gene is associated with Muenke syndrome? | Muenke syndrome has been related to a mutation on the Fibroblast Growth Factor Receptor (FGFR3) gene. | Hypochondroplasia (HCH) and Muenke syndrome (MS) are caused by mutations on
FGFR3 gene. FGFR3 is known to play a role in controlling nervous system
development. We describe the clinical and neuroradiological findings of the
first two patients, to our knowledge, affected by HCH and MS, respectively, in
whom bilateral dysgenesis of the medial temporal lobe structures has been
observed. In both patients diagnosis was confirmed by molecular analysis. They
were mentally normal and showed similarities in early-onset temporal
lobe-related seizures. In both patients EEG recorded bilateral temporal region
discharges. MRI detected temporal lobe anomalies with inadequate differentiation
between white and gray matter, defective gyri, and abnormally shaped
hippocampus. BACKGROUND: Muenke syndrome is a genetically determined craniosynostosis that
involves one or both coronal sutures. In some patients it is associated with
skeletal abnormalities such as thimble-like middle phalanges, coned epiphysis,
and/or neurological impairment, namely sensorineural hearing loss or mental
retardation. In spite of a variable phenotype, Muenke syndrome has been related
to a unique mutation on the FGFR3 gene, Pro 250 to Arg, which is characteristic
of this disease. Because of the incomplete penetrance of this anomaly, the
suspicion of Muenke syndrome must be raised in any child with uni- or bilateral
coronal craniosynostosis, and the genetic analysis propounded even in the
absence of extracranial features.
ILLUSTRATIVE CASES: We report the cases of two sisters who presented with Muenke
syndrome and whose affected mother did not have any form of craniosynostosis. Muenke syndrome, also known as FGFR3-associated coronal synostosis, is defined
molecularly by the presence of a heterozygous nucleotide transversion, c.749C>G,
encoding the amino acid substitution Pro250Arg, in the fibroblast growth factor
receptor type 3 gene (FGFR3). This frequently occurs as a new mutation,
manifesting one of the highest documented rates for any transversion in the
human genome. To understand the biology of this mutation, we have investigated
its parental origin, and the ages of the parents, in 19 families with de novo
c.749C>G mutations. All ten informative cases originated from the paternal
allele (95% confidence interval 74-100% paternal); the average paternal age at
birth overall was 34.7 years. An exclusive paternal origin of mutations, and
increased paternal age, were previously described for a different mutation
(c.1138G>A) of the FGFR3 gene causing achondroplasia, as well as for mutations
of the related FGFR2 gene causing Apert, Crouzon and Pfeiffer syndromes. We
conclude that similar biological processes are likely to shape the occurrence of
this c.749C>G mutation as for other mutations of FGFR3 as well as FGFR2. P250R mutation in the FGFR3 gene also known as Muenke syndrome is associated
with coronal craniosynostosis, sensorineural deafness, craniofacial, and digital
abnormalities. We report a family with this mutation associated with sudden
death in an affected newborn, most probably due to upper airway obstruction. Muenke syndrome is an autosomal domit disorder characterized by coronal
suture craniosynostosis, hearing loss, developmental delay, carpal and tarsal
fusions, and the presence of the Pro250Arg mutation in the FGFR3 gene. Reduced
penetrance and variable expressivity contribute to the wide spectrum of clinical
findings in Muenke syndrome. To better define the clinical features of this
syndrome, we initiated a study of the natural history of Muenke syndrome. To
date, we have conducted a standardized evaluation of nine patients with a
confirmed Pro250Arg mutation in FGFR3. We reviewed audiograms from an additional
13 patients with Muenke syndrome. A majority of the patients (95%) demonstrated
a mild-to-moderate, low frequency sensorineural hearing loss. This pattern of
hearing loss was not previously recognized as characteristic of Muenke syndrome.
We also report on feeding and swallowing difficulties in children with Muenke
syndrome. Combining 312 reported cases of Muenke syndrome with data from the
nine NIH patients, we found that females with the Pro250Arg mutation were
significantly more likely to be reported with craniosynostosis than males (P <
0.01). Based on our findings, we propose that the clinical management should
include audiometric and developmental assessment in addition to standard
clinical care and appropriate genetic counseling. Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in
fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of
craniosynostosis in humans. We have used gene targeting to introduce the Muenke
syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded
skull and shortened snout (often skewed) with dental malocclusion was observed
in a minority of heterozygotes and many homozygotes. Development of this
incompletely penetrant skull phenotype was dependent on genetic background and
sex, with males more often affected. However, these cranial abnormalities were
rarely attributable to craniosynostosis, which was only present in 2/364
mutants; more commonly, we found fusion of the premaxillary and/or zygomatic
sutures. We also found decreased cortical thickness and bone mineral densities
in long bones. We conclude that although both cranial and long bone development
is variably affected by the murine Fgfr3(P244R) mutation, coronal
craniosynostosis is not reliably reproduced. Mutations in the gene that encodes Fibroblast Growth Factor Receptor 3 (FGFR3)
are associated with Achondroplasia (MIM 100800), Hypochondroplasia (MIM 146000),
Muenke Syndrome (MIM 602849), Thanatophoric Dysplasia (MIM 187600, MIM 187601)
and Lacrimo-Auriculo-Dento-Digital Syndrome (MIM 149730).Here we report a
clinical and molecular study in a large cohort of 125 Portuguese patients with
these skeletal disorders. The identification of the P250R mutation allowed the
confirmation of the Muenke Syndrome in 9 out of the 52 cases referred. Two known
mutations were found in the Thanatophoric Dysplasia referred cases. No mutations
were identified in the LADD syndrome patient. In Achondroplasia and
Hypochondroplasia, genetic heterogeneity was present amongst the 70 clinically
diagnosed patients with 5 different mutations identified. As in other studies,
complex phenotypic heterogeneity amongst patients carrying the same gene defect
was observed. In several cases, the new amino acids encoded, as a consequence of
mutations, were related to the severity of patients' phenotype. The presence of
10 misdiagnosed cases emphasizes the importance of performing mutation analysis
of the hotspot regions responsible for both dysplasias (Ach and Hch). For
patients with an unquestionable clinical diagnosis, lacking the most common
mutations, a complete screening of FGFR3 is necessary. Muenke syndrome (MS), also known as Muenke nonsyndromic coronal
craniosynostosis, is an autosomal domit condition which can be distinguished
from the more common forms of acrocephalosyndactyly but presents a significant
variable phenotype. We report on a set of identical twins with a de novo C749G
mutation in the FGFR3 gene codon 250 after a pregcy complicated by prenatal
exposure to Nortriptyline. These patients illustrate the variable expressivity
of MS in association with an identical gene mutation. The Muenke syndrome (MS) is characterized by unicoronal or bicoronal
craniosynostosis, midfacial hypoplasia, ocular hypertelorism, and a variety of
minor abnormalities associated with a mutation in the fibroblast growth factor
receptor 3 (FGFR3) gene. The birth prevalence is approximately one in 10,000
live births, accounting for 8-10% of patients with coronal synostosis. Although
MS is a relatively common diagnosis in patients with craniosynostosis syndromes,
with autosomal domit inheritance, there has been no report of MS, in an
affected Korean family with typical cephalo-facial morphology that has been
confirmed by molecular studies. Here, we report a familial case of MS in a
female patient with a Pro250Arg mutation in exon 7 (IgII-IGIII linker domain) of
the FGFR3 gene. This patient had mild midfacial hypoplasia, hypertelorism,
downslanting palpebral fissures, a beak shaped nose, plagio-brachycephaly, and
mild neurodevelopmental delay. The same mutation was confirmed in the patient's
mother, two of the mother's sisters and the maternal grandfather. The severity
of the cephalo-facial anomalies was variable among these family members. In about 30% of the patients with syndromal craniosynostosis, a genetic mutation
can be traced. For the purpose of adequate genetic counseling and treatment of
these patients, the full spectrum of clinical findings for each specific
mutation needs to be appreciated. The Pro250Arg mutation in the FGFR3 gene is
found in patients with Muenke syndrome and is one of the most frequently
encountered mutations in craniosynostosis syndromes. A number of studies on the
relationship between genotype and phenotype concerning this specific mutation
have been published. Two Dutch families with Muenke syndrome were screened for
the reported characteristics of this syndrome and for additional features. New
phenotypical findings were hypoplasia of the frontal sinus, ptosis of the upper
eyelids, dysplastic elbow joints with restricted elbow motion, and mild
cutaneous syndactyly. Incidentally, polydactyly, severe ankylosis of the elbow,
fusion of cervical vertebrae, and epilepsy were found. Upper eyelid ptosis is
thought to be pathognomonic for Saethre-Chotzen syndrome but was also observed
in our series of patients with Muenke syndrome. Because Muenke and
Saethre-Chotzen syndrome can have similar phenotypes, DNA analysis is needed to
distinguish between these syndromes, even when a syndrome diagnosis is already
made in a family member. Muenke syndrome is characterized by various craniofacial deformities and is
caused by an autosomal-domit activating mutation in fibroblast growth factor
receptor 3 (FGFR3(P250R) ). Here, using mice carrying a corresponding mutation
(FgfR3(P244R) ), we determined whether the mutation affects temporomandibular
joint (TMJ) development and growth. In situ hybridization showed that FgfR3 was
expressed in condylar chondroprogenitors and maturing chondrocytes that also
expressed the Indian hedgehog (Ihh) receptor and transcriptional target Patched
1(Ptch1). In FgfR3(P244R) mutants, the condyles displayed reduced levels of Ihh
expression, H4C-positive proliferating chondroprogenitors, and collagen type II-
and type X-expressing chondrocytes. Primary bone spongiosa formation was also
disturbed and was accompanied by increased osteoclastic activity and reduced
trabecular bone formation. Treatment of wild-type condylar explants with
recombit FGF2/FGF9 decreased Ptch1 and PTHrP expression in
superficial/polymorphic layers and proliferation in chondroprogenitors. We also
observed early degenerative changes of condylar articular cartilage, abnormal
development of the articular eminence/glenoid fossa in the TMJ, and fusion of
the articular disc. Analysis of our data indicates that the activating
FgfR3(P244R) mutation disturbs TMJ developmental processes, likely by reducing
hedgehog signaling and endochondral ossification. We suggest that a balance
between FGF and hedgehog signaling pathways is critical for the integrity of TMJ
development and for the maintece of cellular organization. Muenke syndrome is an autosomal domit craniosynostosis syndrome resulting
from a defining point mutation in the Fibroblast Growth Factor Receptor3 (FGFR3)
gene. Muenke syndrome is characterized by coronal craniosynostosis (bilateral
more often than unilateral), hearing loss, developmental delay, and carpal
and/or tarsal bone coalition. Tarsal coalition is a distinct feature of Muenke
syndrome and has been reported since the initial description of the disorder in
the 1990s. Although talocalcaneal coalition is the most common tarsal coalition
in the general population, it has never previously been reported in a patient
with Muenke syndrome. We present a 7-year-old female patient with Muenke
syndrome and symptomatic talocalcaneal coalition. She presented at the age of 7
with limping, tenderness and pain in her right foot following a fall and strain
of her right foot. She was treated with ibuprofen, shoe inserts, a CAM walker
boot, and stretching exercises without much improvement in symptoms. A computed
tomography (CT) scan revealed bilateral talocalcaneal coalitions involving the
middle facet. She underwent resection of the talocalcaneal coalitions, remaining
pain-free post-operatively with an improvement in her range of motion, gait, and
mobility. This report expands the phenotype of tarsal coalition in Muenke
syndrome to include talocalcaneal coalition. A literature review revealed a high
incidence of tarsal coalition in all FGFR related craniosynostosis syndromes
when compared to the general population, a difference that is statistically
significant. The most common articulation involved in all syndromic
craniosynostoses associated with FGFR mutations is the calcaneocuboid
articulation. Muenke is a fibroblast growth factor receptor 3 (FGFR-3)-associated syndrome,
which was first described in late 1990 s. Muenke syndrome is an autosomal
domit disorder characterized mainly by coronal suture craniosynostosis,
hearing impairment and intellectual disability. The syndrome is defined
molecularly by a unique point mutation c.749C > G in exon 7 of the FGFR3 gene
which results to an amino acid substitution p.Pro250Arg of the protein product.
Despite the fact that the mutation rate at this nucleotide is one of the most
frequently described in human genome, few Muenke familial case reports are
published in current literature. We describe individuals among three generations
of a Greek family who are carriers of the same mutation. Medical record and
physical examination of family members present a wide spectrum of clinical
manifestations. In particular, a 38-year-old woman and her father appear milder
clinical findings regarding craniofacial characteristics compared to her uncle
and newborn female child. This familial case illustrates the variable
expressivity of Muenke syndrome in association with an identical gene mutation. |
Is it safe to use Abatacept during pregnancy? | It is not safe to use the drug abatacept during pregnancy, since there is very limited experience/knowledge yet. Additionally, it is recommended to withdraw the drug before pregnancy. | A consensus paper concerning the interaction of anti-rheumatic drugs and
reproduction was published in 2006, representing data collected during the year
2004 and 2005. Because of an increasing use of biological agents in women of
fertile age, the information was updated for the years 2006 and 2007. Experts
disagree whether TNF-inhibitors should be stopped as soon as pregcy is
recognized or may be continued throughout pregcy. Pregcy experience with
abatacept and rituximab is still too limited to prove their safety for the
developing fetus. They must be withdrawn before a planned pregcy. LEF has not
been proven to be a human teratogen. Registries of transplant recipients have
shown that cyclosporin (CsA) and tacrolimus do not increase the rate of
congenital anomalies, whereas mycophenolate mofetil (MMF) clearly carries a risk
for congenital anomalies. Prophylactic withdrawal of drugs before pregcy is
mandatory for abatacept, rituximab, LEF and MMF. Data remain insufficient for
gonadal toxicity of immunosuppressive drugs in men and for excretion of these
drugs in human breast milk. During pregcy, oestrogen and progesterone levels are increased. Consequently
the initial predomit immune cellular response (Th1 type) is decreased,
whereas humoral response (Th2 type) is increased. Due to this switch, a lot of
Th2 anti-inflammatory cytokines IL-4 and IL-10 are synthesized. During the last
months of pregcy Treg lymphocytes level is elevated leading to overexpression
of IL-4 and IL-10. Due to these mechanisms, reduce disease activity of
rheumatoid arthritis (RA) occurred. Impaired fertility has not been demonstrated
in women with RA. However, some studies suggest that polyarthritis could induced
a reduced weight at birth and more frequent pregcy and delivery
complications. Methotrexate and biotherapies have demonstrated no effect on
fertility; however these drugs must be stopped before conception for a period
equal to seven fold of the half live of the molecule. No teratogenic effect are
known for sulfazalasine and hydroxychloroquine; these drugs could be used during
pregcy. It is also the same for ciclosporine, which used is quite unfrequent
in RA. Methotrexate is teratogenic in animal models and is forbidden during
pregcy. For leflunomide which is metabolised in A771726, highly teratogenic,
a washout period of 3,5 months is necessary. All commercially available TNFalpha
inhibitors are classified by the food and Drug Administration as pregcy risk
category B: no adverse pregcy adverse effects have been observed in animal
studies, but there have been insufficient controlled human studies. The
published experiences with TNFalpha inhibition in pregcy is limited to some
case reports and ongoing registry. More recently some cases of Vater syndromes
(polymalformations) were possibly related to TNFalpha blocking agents. Such
treatment must be avoided during pregcy. Only few case reports are published
concerning rituximab use during pregcy. No data have been found for
abatacept. There are a wide variety of medications available to treat patients with
rheumatoid arthritis, many of which are considered unsafe during pregcy. It
is important to tailor a treatment regimen that stabilises the woman's disease
prior to conception, using medications that are safe to continue throughout
pregcy and the post-partum period. Drugs that may be safely used during
pregcy include NSAIDs, corticosteroids, plus several DMARDs, including
sulfasalazine and hydroxychloroquine. Drugs recommended to be stopped before
pregcy include methotrexate and leflunomide, plus the biologics: anti-TNF
agents, rituximab and abatacept. |
What is DeepCAGE? | The cap analysis of gene expression (CAGE) technology has been established to detect transcriptional starting sites (TSSs) and expression levels by utilizing 5' cDNA tags and PCR. It has been reported that the amount of templates is proportional to the amplification efficiency of PCR. CAGE has been used as a key technique for analyzing promoter activity and finding new transcripts including alternative spliced products and noncoding transcripts. DeepCAGE can be utilized for high-throughput next-generation sequencing technology. DeepCAGE can produce much deeper transcriptome datasets and can reveal more details of the regulatory network. | Finding and characterizing mRNAs, their transcription start sites (TSS), and
their associated promoters is a major focus in post-genome biology. Mammalian
cells have at least 5-10 magnitudes more TSS than previously believed, and
deeper sequencing is necessary to detect all active promoters in a given tissue.
Here, we present a new method for high-throughput sequencing of 5' cDNA
tags-DeepCAGE: merging the Cap Analysis of Gene Expression method with
ultra-high-throughput sequence technology. We apply DeepCAGE to characterize 1.4
million sequenced TSS from mouse hippocampus and reveal a wealth of novel core
promoters that are preferentially used in hippocampus: This is the most
comprehensive promoter data set for any tissue to date. Using these data, we
present evidence indicating a key role for the Arnt2 transcription factor in
hippocampus gene regulation. DeepCAGE can also detect promoters used only in a
small subset of cells within the complex tissue. |
Is STAT3 involved in EIF2AK2-dependent suppression of autophagy? | STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy | In a screen designed to identify novel inducers of autophagy, we discovered that
STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic
inhibition of STAT3 stimulated autophagy in vitro and in vivo, while
overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable,
and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain
of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2
EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic
inhibition of STAT3 stimulated the activating phosphorylation of PKR and
consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited
autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like
palmitate. STAT3-targeting chemicals and palmitate caused the disruption of
inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α
phosphorylation, which facilitates autophagy induction. These results unravel an
unsuspected mechanism of autophagy control that involves STAT3 and PKR as
interacting partners. A chemical screen designed to identify novel inducers of autophagy led to the
discovery that signal transducer and activator of transcription 3 (STAT3)
inhibitors can potently stimulate the autophagic flux. Although STAT3 is best
known as a pro-inflammatory and oncogenic transcription factor, mechanistic
analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear,
pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic
translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase
2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA.
This interaction, which could be recapitulated using recombit proteins in
pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the
SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus,
STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or
genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation
and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of
STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the
nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with
EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e.,
Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand
RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the
inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter
does so in cell-free systems in vitro. A further screen designed to identify
EIF2AK2-dependent autophagy inducers revealed that several fatty acids including
palmitate trigger autophagy via a pathway that involves the disruption of the
STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated
protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These
results reveal an unsuspected crosstalk between cellular metabolism (fatty
acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and
translational control (EIF2S1) that regulates autophagy. |
RTS S AS01 vaccine was developed to prevent which disease? | RTS,S/AS01 vaccine was developed for prevention of malaria. | The RTS,S/AS01(E) malaria vaccine candidate has recently entered Phase 3
testing. Reaching this important milestone is the culmination of more than 20
years of research and development by GlaxoSmithKline and partners and
collaborators. The vaccine has been developed to protect young children and
infants living in Sub-Saharan Africa against clinical and severe disease caused
by Plasmodium falciparum infection. Over the past 9 years, RTS,S/AS has been
evaluated in multiple Phase 2 studies. The vaccine was shown to have a favorable
safety profile and to be well tolerated in all age groups in which it was
tested, including the intended target population of infants and young children
in Sub-Saharan Africa. Data obtained so far suggest that RTS,S/AS can be
co-administered with other vaccines included in the routine Expanded Program of
Immunization (EPI). In Phase 2 testing, the vaccine candidate was shown to
confer significant protection against P. falciparum infection and clinical
disease, including severe malaria. Furthermore, a trend towards an indirect
beneficial effect of the vaccine on non-malarial morbidities has been observed
in several trials. In this paper, we will describe the genesis of the RTS,S/AS
concept, including the rationale for selecting the circumsporozoite protein
(CSP) as the target antigen. Early development history of the vaccine will be
briefly described. We will present the most salient results from recent Phase 2
studies conducted in the target pediatric population, which have led to the
decision to progress RTS,S/AS to Phase 3 testing. If the Phase 3 results confirm
the observations made during Phase 2 testing, the RTS,S/AS vaccine, when broadly
implemented and judiciously integrated with other malaria-prevention measures,
would have a major public-health impact in Sub-Saharan Africa. BACKGROUND: The malaria vaccine candidate antigen RTS,S includes parts of the
pre-erythrocytic stage circumsporozoite protein fused to the Hepatitis B surface
antigen. Two Adjuvant Systems are in development for this vaccine, an oil-in
water emulsion--based formulation (AS02) and a formulation based on liposomes
(AS01).
METHODS & PRINCIPAL FINDINGS: In this Phase II, double-blind study
(NCT00307021), 180 healthy Gabonese children aged 18 months to 4 years were
randomized to receive either RTS,S/AS01(E) or RTS,S/AS02(D), on a 0-1-2 month
vaccination schedule. The children were followed-up daily for six days after
each vaccination and monthly for 14 months. Blood samples were collected at 4
time-points. Both vaccines were well tolerated. Safety parameters were
distributed similarly between the two groups. Both vaccines elicited a strong
specific immune response after Doses 2 and 3 with a ratio of anti-CS GMT titers
(AS02(D)/AS01(E)) of 0.88 (95% CI: 0.68-1.15) post-Dose 3. After Doses 2 and 3
of experimental vaccines, anti-CS and anti-HBs antibody GMTs were higher in
children who had been previously vaccinated with at least one dose of hepatitis
B vaccine compared to those not previously vaccinated.
CONCLUSIONS: RTS,S/AS01(E) proved similarly as well tolerated and immunogenic as
RTS,S/AS02(D), completing an essential step in the age de-escalation process
within the RTS,S clinical development plan.
TRIAL REGISTRATION: ClinicalTrials.gov. NCT00307021. BACKGROUND: The RTS,S/AS01(E) malaria candidate vaccine is being developed for
immunization of African infants through the Expanded Program of Immunization
(EPI).
METHODS: This phase 2, randomized, open, controlled trial conducted in Ghana,
Tanzania, and Gabon evaluated the safety and immunogenicity of RTS,S/AS01(E)
when coadministered with EPI vaccines. Five hundred eleven infants were
randomized to receive RTS,S/AS01(E) at 0, 1, and 2 months (in 3 doses with
diphtheria, tetanus, and whole-cell pertussis conjugate [DTPw]; hepatitis B
[HepB]; Haemophilus influenzae type b [Hib]; and oral polio vaccine [OPV]),
RTS,S/AS01(E) at 0, 1, and 7 months (2 doses with DTPwHepB/Hib+OPV and 1 dose
with measles and yellow fever), or EPI vaccines only.
RESULTS: The occurrences of serious adverse events were balanced across groups;
none were vaccine-related. One child from the control group died. Mild to
moderate fever and diaper dermatitis occurred more frequently in the
RTS,S/AS01(E) coadministration groups. RTS,S/AS01(E) generated high
anti-circumsporozoite protein and anti-hepatitis B surface antigen antibody
levels. Regarding EPI vaccine responses upon coadministration when considering
both immunization schedules, despite a tendency toward lower geometric mean
titers to some EPI antigens, predefined noninferiority criteria were met for all
EPI antigens except for polio 3 when EPI vaccines were given with RTS,S/AS01(E)
at 0, 1, and 2 months. However, when antibody levels at screening were taken
into account, the rates of response to polio 3 antigens were comparable between
groups.
CONCLUSION: RTS,S/AS01(E) integrated in the EPI showed a favorable safety and
immunogenicity evaluation. Trial registration. ClinicalTrials.gov identifier:
NCT00436007 . GlaxoSmithKline study ID number: 106369 (Malaria-050). BACKGROUND: The RTS,S/AS01(E) candidate malaria vaccine is being developed for
immunisation of infants in Africa through the expanded programme on immunisation
(EPI). 8 month follow-up data have been reported for safety and immunogenicity
of RTS,S/AS01(E) when integrated into the EPI. We report extended follow-up to
19 months, including efficacy results.
METHODS: We did a randomised, open-label, phase 2 trial of safety and efficacy
of the RTS,S/AS01(E) candidate malaria vaccine given with EPI vaccines between
April 30, 2007, and Oct 7, 2009, in Ghana, Tanzania, and Gabon. Eligible
children were 6-10 weeks of age at first vaccination, without serious acute or
chronic illness. All children received the EPI diphtheria, tetanus, pertussis
(inactivated whole-cell), and hepatitis-B vaccines, Haemophilus influenzae type
b vaccine, and oral polio vaccine at study months 0, 1, and 2, and measles
vaccine and yellow fever vaccines at study month 7. Participants were randomly
assigned (1:1:1) to receive three doses of RTS,S/AS01(E) at 6, 10, and 14 weeks
(0, 1, 2 month schedule) or at 6 weeks, 10 weeks, and 9 months (0, 2, 7 month
schedule) or placebo. Randomisation was according to a predefined block list
with a computer-generated randomisation code. Detection of serious adverse
events and malaria was by passive case detection. Antibodies against Plasmodium
falciparum circumsporozoite protein and HBsAg were monitored for 19 months. This
study is registered with ClinicalTrials.gov, number NCT00436007.
FINDINGS: 511 children were enrolled. Serious adverse events occurred in 57
participants in the RTS,S/AS01(E) 0, 1, 2 month group (34%, 95% CI 27-41), 47 in
the 0, 1, 7 month group (28%, 21-35), and 49 (29%, 22-36) in the control group;
none were judged to be related to study vaccination. At month 19,
anticircumsporozoite immune responses were significantly higher in the
RTS,S/AS01(E) groups than in the control group. Vaccine efficacy for the 0, 1, 2
month schedule (2 weeks after dose three to month 19, site-adjusted
according-to-protocol analysis) was 53% (95% CI 26-70; p=0·0012) against first
malaria episodes and 59% (36-74; p=0·0001) against all malaria episodes. For the
entire study period, (total vaccinated cohort) vaccine efficacy against all
malaria episodes was higher with the 0, 1, 2 month schedule (57%, 95% CI 33-73;
p=0·0002) than with the 0, 1, 7 month schedule (32% CI 16-45; p=0·0003). 1 year
after dose three, vaccine efficacy against first malaria episodes was similar
for both schedules (0, 1, 2 month group, 61·6% [95% CI 35·6-77·1], p<0·001; 0,
1, 7 month group, 63·8% [40·4-78·0], p<0·001, according-to-protocol cohort).
INTERPRETATION: Vaccine efficacy was consistent with the target put forward by
the WHO-sponsored malaria vaccine technology roadmap for a first-generation
malaria vaccine. The 0, 1, 2 month vaccine schedule has been selected for phase
3 candidate vaccine assessment.
FUNDING: Program for Appropriate Technology in Health Malaria Vaccine
Initiative; GlaxoSmithKline Biologicals. BACKGROUND: There has been much debate about the appropriate statistical
methodology for the evaluation of malaria field studies and the challenges in
interpreting data arising from these trials.
METHODS: The present paper describes, for a pivotal phase III efficacy of the
RTS, S/AS01 malaria vaccine, the methods of the statistical analysis and the
rationale for their selection. The methods used to estimate efficacy of the
primary course of vaccination, and of a booster dose, in preventing clinical
episodes of uncomplicated and severe malaria, and to determine the duration of
protection, are described. The interpretation of various measures of efficacy in
terms of the potential public health impact of the vaccine is discussed.
CONCLUSIONS: The methodology selected to analyse the clinical trial must be
scientifically sound, acceptable to regulatory authorities and meaningful to
those responsible for malaria control and public health policy. BACKGROUND: An effective malaria vaccine, deployed in conjunction with other
malaria interventions, is likely to substantially reduce the malaria burden.
Efficacy against severe malaria will be a key driver for decisions on
implementation. An initial study of an RTS, S vaccine candidate showed promising
efficacy against severe malaria in children in Mozambique. Further evidence of
its protective efficacy will be gained in a pivotal, multi-centre, phase III
study. This paper describes the case definitions of severe malaria used in this
study and the programme for standardized assessment of severe malaria according
to the case definition.
METHODS: Case definitions of severe malaria were developed from a literature
review and a consensus meeting of expert consultants and the RTS, S Clinical
Trial Partnership Committee, in collaboration with the World Health Organization
and the Malaria Clinical Trials Alliance. The same groups, with input from an
Independent Data Monitoring Committee, developed and implemented a programme for
standardized data collection.The case definitions developed reflect the typical
presentations of severe malaria in African hospitals. Markers of disease
severity were chosen on the basis of their association with poor outcome,
occurrence in a significant proportion of cases and on an ability to standardize
their measurement across research centres. For the primary case definition, one
or more clinical and/or laboratory markers of disease severity have to be
present, four major co-morbidities (pneumonia, meningitis, bacteraemia or
gastroenteritis with severe dehydration) are excluded, and a Plasmodium
falciparum parasite density threshold is introduced, in order to maximize the
specificity of the case definition. Secondary case definitions allow inclusion
of co-morbidities and/or allow for the presence of parasitaemia at any density.
The programmatic implementation of standardized case assessment included a
clinical algorithm for evaluating seriously sick children, improvements to care
delivery and a robust training and evaluation programme for clinicians.
CONCLUSIONS: The case definition developed for the pivotal phase III RTS, S
vaccine study is consistent with WHO recommendations, is locally applicable and
appropriately balances sensitivity and specificity in the diagnosis of severe
malaria. Processes set up to standardize severe malaria data collection will
allow robust assessment of the efficacy of the RTS, S vaccine against severe
malaria, strengthen local capacity and benefit patient care for subjects in the
trial.
TRIAL REGISTRATION: Clinicaltrials.gov NCT00866619. BACKGROUND: A pivotal phase III study of the RTS,S/AS01 malaria candidate
vaccine is ongoing in several research centres across Africa. The development
and establishment of quality systems was a requirement for trial conduct to meet
international regulatory standards, as well as providing an important capacity
strengthening opportunity for study centres.
METHODS: Standardized laboratory methods and quality assurance processes were
implemented at each of the study centres, facilitated by funding partners.
RESULTS: A robust protocol for determination of parasite density based on actual
blood cell counts was set up in accordance with World Health Organization
recommendations. Automated equipment including haematology and biochemistry
analyzers were put in place with standard methods for bedside testing of
glycaemia, base excess and lactacidaemia. Facilities for X-rays and basic
microbiology testing were also provided or upgraded alongside health care
infrastructure in some centres. External quality assurance assessment of all
major laboratory methods was established and method qualification by each
laboratory demonstrated. The resulting capacity strengthening has ensured
laboratory evaluations are conducted locally to the high standards required in
clinical trials.
CONCLUSION: Major efforts by study centres, together with support from
collaborating parties, have allowed standardized methods and robust quality
assurance processes to be put in place for the phase III evaluation of the RTS,
S/AS01 malaria candidate vaccine. Extensive training programmes, coupled with
continuous commitment from research centre staff, have been the key elements
behind the successful implementation of quality processes. It is expected these
activities will culminate in healthcare benefits for the subjects and
communities participating in these trials.
TRIAL REGISTRATION: Clinicaltrials.gov NCT00866619. Malaria remains one of the few diseases those continue to scourge human
civilization despite the significant advances in disease control strategies over
the last century. Malaria is responsible for more than 500 million cases and 1-3
million deaths annually. Approximately 85% of these deaths are among children,
mostly in Africa, primarily due to P. falciparum. Whole cell vaccines,
irradiated sporozoites and genetically attenuated sporozoites have demonstrated
long lasting, sterile protection against plasmodium infection in animal and
experimental clinical studies. Atypical membrane protein 1 and merozoite surface
protein 1 are the two most extensively studied asexual blood stage vaccine
candidates. The most promising candidate vaccine under development is RTS, S
combined with AS01 adjuvant. Initial results from phase III trials of this
candidate vaccine show 50% reduction of malaria in 5-17 mo aged children during
the 12 mo after vaccination. WHO anticipates that the RTS,S/AS01 vaccine will be
recommended for the 6-14 week age group for co-administration together with
other vaccines as part of routine immunization programs in malaria endemic
countries. Malaria vaccine could play an important role in elimination and
eventual eradication of malaria. The Malaria Policy Advisory Committee to the World Health Organization met in
Geneva, Switzerland from 13 to 15 March, 2013. This article provides a summary
of the discussions, conclusions and recommendations from that meeting.Meeting
sessions included: a review of the efficacy of artemisinin-based combination
therapy in Guyana and Suriname; the outcomes from a consultation on non-malaria
febrile illness; the outcomes from the second meeting of the Evidence Review
Group on malaria burden estimation; an update on the review of the WHO
Guidelines for the Treatment of Malaria; an update regarding progress on the
constitution of the vector control Technical Expert Group; updates on the RTS,
S/AS01 vaccine and the malaria vaccine technology roadmap; ficing and
resource allocation for malaria control; malaria surveillance and the need for a
surveillance, monitoring and evaluation Technical Expert Group; criteria and
classification related to malaria elimination; the next meeting of the Evidence
Review Group on Intermittent Preventive Treatment in pregcy; an update on the
soon-to-be launched Elimination Scenario Planning Tool; and an update on the
process for the Global Technical Strategy for Malaria Control and Elimination
(2016-2025).Policy statements, position statements, and guidelines that arise
from the MPAC meeting conclusions and recommendations will be formally issued
and disseminated to World Health Organization Member States by the World Health
Organization Global Malaria Programme. Malaria, which is the result of Plasmodium falciparum infection, is a global
health threat that resulted in 655,000 deaths and 216 million clinical cases in
2010 alone. Recent phase 3 trials with malaria vaccine candidate RTS,S/AS01
(RTS,S) in children has demonstrated modest efficacy against clinical and severe
malaria. RTS,S targets the pre-erythrocytic phase of the disease and induces
high antibody titers against the P. falciparum circumsporozoite protein (CSP)
and a moderate CD4(+) T cell response. The individual contribution of these
adaptive immune responses to protection from infection remains unknown. Here, we
found that prophylactic administration of anti-CSP mAbs derived from an
RTS,S-vaccinated recipient fully protected mice with humanized livers from i.v.-
and mosquito bite–delivered P. falciparum sporozoite challenge. Titers of
anti-CSP that conveyed full protection were within the range observed in human
RTS,S vaccine recipients. Increasing anti-CSP titers resulted in a
dose-dependent reduction of the liver parasite burden. These data indicate that
RTS,S-induced antibodies are protective and provide sterilizing immunity against
P. falciparum infection when reaching or exceeding a critical plasma
concentration. Malaria is one of the most devastating infectious diseases in the developing
world. Until now, only one candidate malaria vaccine RTS,S/AS01 has shown modest
protection in phase 3 trial in African infants. Hence the treatment of malaria
still depends on the current chemotherapeutic drugs. Considering the resistance
of malaria parasites to almost all used antimalarial drugs, aiming at
multi-targets rather than a single target will be a more promising strategy.
Previous studies have shown that myricetin and fisetin exhibited in vitro
antimalarial activity against Plasmodium falciparum, but very little research
focused on the molecular mechanism for their parasiticidal activity. The
cysteine protease falcipain-2 and aspartic protease plasmepsin II have long been
considered as important antimalarial drug targets, especially combined
inhibition of these two proteases. In this study, we determined that myricetin
and fisetin are dual inhibitors of falcipain-2 and plasmepsin II, which might
account for their antimalarial properties. Overall, the dual inhibition of
falcipain-2 and plasmepsin II by myricetin and fisetin has shed light on a
possible mechanism for their antimalarial activity and provided a rationale for
further development as antimalarial drugs. Conflict of interest statement: The trial was sponsored by GlaxoSmithKline
Biologicals SA (GSK), the vaccine developer and manufacturer, and funded by both
GSK Biologicals SA and the PATH Malaria Vaccine Initiative (MVI). All centers
declare receiving a grant from MVI for running the trial. Author travel and
accommodation related to this trial were ficed by MVI. GlaxoSmithKline
Biologicals SA received a grant from MVI to run the trial. MVI received a grant
from the Bill & Melinda Gates Foundation to run this trial and to compensate MVI
authors for trial-related travel. Additional conflicts of interest are as
follows: JJA and PAl declare that their institutions received grant from the
Catalan government and from the International Agency for Development and
Cooperation. NA, CO, and KO declare that their institutions received a grant
from the Malaria Clinical Trial Alliance. PB, SD, BG, CK, PL, CMai, GMwam, BO,
and LO declare that their institution has received grants from MVI for other
malaria studies. KM declares that his institution received a grant from the
Wellcome Trust and that he received support from USAID and the Bill & Melinda
Gates Foundation to participate in a scientific advisory group on malaria. MML
declares that she received non-ficial support from the WHO and the Biomérieux
Foundation. PN declares that she received ficial support from GSK to present
the results of the study at ASTMH congress in 2012. LO declares that he received
support from GSK to carry out clinical and epidemiological studies. JSa has
received (for the Center) some GlaxoSmithKline group of companies' consultancy
fees for other studies. MTa is a board member of the Optimus Foundation, and his
institution is reimbursed for his activities on the scientific advisory board of
the Novartis Institute for Tropical Diseases. He also has received for his
institution other grants from MVI and from the Bill & Melinda Gates Foundation,
and travel reimbursements from MVI and Sanaria corp. All GSK Vaccines authors
are, or were at the time of the study, employed by the GlaxoSmithKline group of
companies. JC now works as an independent consultant for GSK Vaccines. WRB, JC,
EJ, DLa, OOA, JV, AL, and MLi have shares/stock options in the GlaxoSmithKline
group of companies. JC and WRB declare that they are named inventors on patents
for which the rights have been assigned to GlaxoSmithKline group of companies.
DK, DLe, and BS are employees at PATH-MVI. DSc is employed by the London School
of Hygiene & Tropical Medicine, and his consultancy activities for MVI are
funded as a grant to the LSHTM by MVI. DK holds stock or stock options from
Merck, Sharpe & Dome. BACKGROUND: For regulatory approval, consistency in manufacturing of vaccine
lots is expected to be demonstrated in confirmatory immunogenicity studies using
two-sided equivalence trials. This randomized, double-blind study (NCT01323972)
assessed consistency of three RTS,S/AS01 malaria vaccine batches formulated from
commercial-scale purified antigen bulk lots in terms of anti-CS-responses
induced.
METHODS: Healthy children aged 5-17 months were randomized (1:1:1:1) to receive
RTS,S/AS01 at 0-1-2 months from one of three commercial-scale purified antigen
bulk lots (1600 litres-fermentation scale; commercial-scale lots), or a
comparator vaccine batch made from pilot-scale purified antigen bulk lot (20
litres-fermentation scale; pilot-scale lot). The co-primary objectives were to
first demonstrate consistency of antibody responses against circumsporozoite
(CS) protein at one month post-dose 3 for the three commercial-scale lots and
second demonstrate non-inferiority of anti-CS antibody responses at one month
post-dose 3 for the commercial-scale lots compared to the pilot-scale lot.
Safety and reactogenicity were evaluated as secondary endpoints.
RESULTS: One month post-dose-3, anti-CS antibody geometric mean titres (GMT) for
the 3 commercial scale lots were 319.6 EU/ml (95% confidence interval (CI):
268.9-379.8), 241.4 EU/ml (207.6-280.7), and 302.3 EU/ml (259.4-352.3).
Consistency for the RTS,S/AS01 commercial-scale lots was demonstrated as the
two-sided 95% CI of the anti-CS antibody GMT ratio between each pair of lots was
within the range of 0.5-2.0. GMT of the pooled commercial-scale lots (285.8
EU/ml (260.7-313.3)) was non-inferior to the pilot-scale lot (271.7 EU/ml
(228.5-323.1)). Each RTS,S/AS01 lot had an acceptable tolerability profile, with
infrequent reports of grade 3 solicited symptoms. No safety signals were
identified and no serious adverse events were considered related to vaccination.
CONCLUSIONS: RTS,S/AS01 lots formulated from commercial-scale purified antigen
bulk batches induced a consistent anti-CS antibody response, and the anti-CS GMT
of pooled commercial-scale lots was non-inferior to that of a lot formulated
from a pilot-scale antigen bulk batch. |
What are the Topological Domains (TADs)? | Topolological domains or TADs are megabase-sized local chromatin interaction domains which are a pervasive structural feature of the genome organization. These domains correlate with regions of the genome that constrain the spread of heterochromatin. The domains are stable across different cell types and highly conserved across species, indicating that topological domains are an inherent property of mammalian genomes. The boundaries of topological domains are enriched for the insulator binding protein CTCF, housekeeping genes, transfer RNAs and short interspersed element (SINE) retrotransposons, indicating that these factors may have a role in establishing the topological domain structure of the genome. | The spatial organization of the genome is intimately linked to its biological
function, yet our understanding of higher order genomic structure is coarse,
fragmented and incomplete. In the nucleus of eukaryotic cells, interphase
chromosomes occupy distinct chromosome territories, and numerous models have
been proposed for how chromosomes fold within chromosome territories. These
models, however, provide only few mechanistic details about the relationship
between higher order chromatin structure and genome function. Recent advances in
genomic technologies have led to rapid advances in the study of
three-dimensional genome organization. In particular, Hi-C has been introduced
as a method for identifying higher order chromatin interactions genome wide.
Here we investigate the three-dimensional organization of the human and mouse
genomes in embryonic stem cells and terminally differentiated cell types at
unprecedented resolution. We identify large, megabase-sized local chromatin
interaction domains, which we term 'topological domains', as a pervasive
structural feature of the genome organization. These domains correlate with
regions of the genome that constrain the spread of heterochromatin. The domains
are stable across different cell types and highly conserved across species,
indicating that topological domains are an inherent property of mammalian
genomes. Finally, we find that the boundaries of topological domains are
enriched for the insulator binding protein CTCF, housekeeping genes, transfer
RNAs and short interspersed element (SINE) retrotransposons, indicating that
these factors may have a role in establishing the topological domain structure
of the genome. |
What is the vibrational theory of olfaction? | The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. The theory proposes that olfactory receptors respond not to the shape of the molecules but to their vibrations. It differs from previous vibrational theories (Dyson, Wright) in providing a detailed and plausible mechanism for biological transduction of molecular vibrations: inelastic electron tunnelling. Thus, the authors, that have proposed this theory, suggest that olfaction, like colour vision and hearing, is a spectral sense. | A novel theory of primary olfactory reception is described. It proposes that
olfactory receptors respond not to the shape of the molecules but to their
vibrations. It differs from previous vibrational theories (Dyson, Wright) in
providing a detailed and plausible mechanism for biological transduction of
molecular vibrations: inelastic electron tunnelling. Elements of the tunnelling
spectroscope are identified in putative olfactory receptors and their associated
G-protein. Means of calculating electron tunnelling spectra of odorant molecules
are described. Several examples are given of correlations between tunnelling
spectrum and odour in structurally unrelated molecules. As predicted, molecules
of very similar shape but differing in vibrations smell different. The most
striking instance is that of pure acetophenone and its fully deuterated analogue
acetophenone-d8, which smell different despite being identical in structure.
This fact cannot, it seems, be explained by structure-based theories of odour.
The evidence presented here suggests instead that olfaction, like colour vision
and hearing, is a spectral sense. The vibrational theory of olfaction assumes that electron transfer occurs across
odorants at the active sites of odorant receptors (ORs), serving as a sensitive
measure of odorant vibrational frequencies, ultimately leading to olfactory
perception. A previous study reported that human subjects differentiated
hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk
compound cyclopentadecanone as evidence supporting the theory. Here, we find no
evidence for such differentiation at the molecular level. In fact, we find that
the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR
expression system and robustly responding to cyclopentadecanone and muscone,
fails to distinguish isotopomers of these compounds in vitro. Furthermore, the
mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other
selected human and mouse ORs, responded similarly to normal, deuterated, and
(13)C isotopomers of their respective ligands, paralleling our results with the
musk receptor OR5AN1. These findings suggest that the proposed vibration theory
does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3,
or other ORs examined. Also, contrary to the vibration theory predictions,
muscone-d30 lacks the 1,380- to 1,550-cm(-1) IR bands claimed to be essential
for musk odor. Furthermore, our theoretical analysis shows that the proposed
electron transfer mechanism of the vibrational frequencies of odorants could be
easily suppressed by quantum effects of nonodorant molecular vibrational modes.
These and other concerns about electron transfer at ORs, together with our
extensive experimental data, argue against the plausibility of the vibration
theory. |
Are CTCF and BORIS involved in genome regulation and cancer? | Yes. CTCF is ubiquitously expressed and plays diverse roles in gene regulation, imprinting, insulation, intra/interchromosomal interactions, nuclear compartmentalisation, and alternative splicing. CTCF has a single paralogue, the testes-specific CTCF-like gene (CTCFL)/BORIS. CTCF and BORIS can be deregulated in cancer. The tumour suppressor gene CTCF can be mutated or deleted in cancer, or CTCF DNA binding can be altered by epigenetic changes. BORIS is aberrantly expressed frequently in cancer, leading some to propose a pro-tumourigenic role for BORIS. However, BORIS can inhibit cell proliferation, and is mutated in cancer similarly to CTCF suggesting BORIS activation in cancer may be due to global genetic or epigenetic changes typical of malignant transformation. | CTCF is a ubiquitous 11 zinc finger (ZF) protein with highly versatile
functions: in addition to transcriptional silencing or activating in a
context-dependent fashion, it organizes epigenetically controlled chromatin
insulators that regulate imprinted genes in soma. Recently, we have identified a
CTCF paralogue, termed BORIS for Brother of the Regulator of Imprinted Sites,
that is expressed only in the testis. BORIS has the same exons encoding the 11
ZF domain as mammalian CTCF genes, and hence interacts with similar cis
elements, but encodes amino and carboxy termini distinct from those in CTCF.
Normally, CTCF and BORIS are expressed in a mutually exclusive pattern that
correlates with re-setting of methylation marks during male germ cell
differentiation. The antagonistic features of these two gene siblings are
underscored by showing that while CTCF overexpression blocks cell proliferation,
expression of BORIS in normally BORIS-negative cells promotes cell growth which
can lead to transformation. The suggestion that BORIS directs epigenetic
reprogramming at CTCF target sites impinges on the observations that human BORIS
is not only abnormally activated in a wide range of human cancers, but also maps
to the cancer-associated amplification region at 20q13. The sibling rivalry
occasioned by aberrant expression of BORIS in cancer may interfere with normal
functions of CTCF including growth suppression, and contribute to epigenetic
dysregulation which is a common feature in human cancer. Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog
with the same central 11Zn fingers (11ZF) that mediate specific interactions
with varying approximately 50-bp target sites. Regulated in vivo occupancy of
such sites may yield structurally and functionally distinct CTCF/DNA complexes
involved in various aspects of gene regulation, including epigenetic control of
gene imprinting and X chromosome inactivation. The latter functions are mediated
by meCpG-sensitive 11ZF binding. Because CTCF is normally present in all somatic
cells, whereas BORIS is active only in CTCF- and 5-methylcytosine-deficient
adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested
to regulate site specificity and timing of epigenetic reprogramming. In addition
to 11ZF-binding paternal imprinting control regions, cancer-testis gene
promoters also undergo remethylation during CTCF/BORIS switching in germ cells.
Only promoters of cancer testis genes are normally silenced in all somatic cells
but activated during spermatogenesis when demethylated in BORIS-positive germ
cells and are found aberrantly derepressed in various tumors. We show here that
BORIS is also expressed in multiple cancers and is thus itself a cancer-testis
gene and that conditional expression of BORIS in normal fibroblasts activates
cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on
regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene,
MAGE-A1. Transition from a hypermethylated/silenced to a
hypomethylated/activated status induced in normal cells by
5-aza-2'-deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and
is associated with complete switching from CTCF to BORIS occupancy at a single
11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding
insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only
few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that
BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of
normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before
treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is
likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target
sequences that mediate induction of cancer-testis genes. Regulatory sequences recognized by the unique pair of paralogous factors, CTCF
and BORIS, have been implicated in epigenetic regulation of imprinting and X
chromosome inactivation. Lung cancers exhibit genome-wide demethylation
associated with derepression of a specific class of genes encoding cancer-testis
(CT) antigens such as NY-ESO-1. CT genes are normally expressed in
BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are
strictly silenced in somatic cells. The present study was undertaken to
ascertain if aberrant activation of BORIS contributes to derepression of
NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that
NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer
cells. Quantitative reverse transcription-PCR analysis revealed robust,
coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but
not normal human bronchial epithelial cells following 5-aza-2'-deoxycytidine
(5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under
clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR,
and chromatin immunoprecipitation (ChIP) experiments showed that induction of
BORIS coincided with direct modulation of chromatin structure within a CpG
island in the 5'-flanking noncoding region of this gene. Cotransfection
experiments using promoter-reporter constructs confirmed that BORIS modulates
NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments
revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike
such sites in the H19-imprinting control region and X chromosome, is insensitive
to CpG methylation in vitro. In vivo occupancy of this site by CTCF was
associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF
to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these
data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter
mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest
that induction of BORIS may be a novel strategy to augment immunogenicity of
pulmonary carcinomas. BORIS, like other members of the 'cancer/testis antigen' family, is normally
expressed in testicular germ cells and repressed in somatic cells, but is
aberrantly activated in cancers. To understand regulatory mechanisms governing
human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE,
we identified three promoters, designated A, B and C, corresponding to
transcription start sites at -1447, -899 and -658 bp upstream of the first ATG.
Alternative promoter usage generated at least five alternatively spliced BORIS
mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis,
BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell
lines tested used only promoter(s) A and/or C while the others utilized
primarily promoters B and C. The differences in promoter usage between normal
and cancer cells suggested that they were subject to differential regulation. We
found that DNA methylation and functional p53 contributes to the negative
regulation of each promoter. Moreover, reduction of CTCF in normally
BORIS-negative human fibroblasts resulted in derepression of BORIS promoters.
These results provide a mechanistic basis for understanding cancer-related
associations between haploinsufficiency of CTCF and BORIS derepression, and
between the lack of functional p53 and aberrant activation of BORIS. BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a
ubiquitously expressed DNA-binding protein with diverse roles in gene expression
and chromatin organisation. BORIS and CTCF have virtually identical zinc finger
domains, yet display major differences in their respective C- and N-terminal
regions. Unlike CTCF, BORIS expression has been reported only in the testis and
certain maligcies, leading to its classification as a "cancer-testis"
antigen. However, the expression pattern of BORIS is both a significant and
unresolved question in the field of DNA binding proteins. Here, we identify
BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells.
We compare the localization of CTCF and BORIS in the nucleus and demonstrate
enrichment of BORIS within the nucleolus, inside the nucleolin core structure
and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF
is not enriched in the nucleolus. Live imaging of cells transiently transfected
with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS
transcript levels are low compared to CTCF, its protein levels are readily
detectable. These findings show that BORIS expression is more widespread than
previously believed, and suggest a role for BORIS in nucleolar function. Cancer germline (CG) genes are normally expressed in germ cells and aberrantly
expressed in a variety of cancers; their immunogenicity has led to the
widespread development of cancer vaccines targeting these antigens. BORIS/CTCFL
is an autosomal CG antigen and promising cancer vaccine target. BORIS is the
only known paralog of CTCF, a gene intimately involved in genomic imprinting,
chromatin insulation, and nuclear regulation. We have previously shown that
BORIS is expressed in epithelial ovarian cancer (EOC) and that its expression
coincides with promoter and global DNA hypomethylation. Recently, 23 different
BORIS mRNA variants have been described, and have been functionally grouped into
six BORIS isoform families (sf1-sf6). In the present study, we have
characterized the expression of BORIS isoform families in normal ovary (NO) and
EOC, the latter of which were selected to include two groups with widely varying
global DNA methylation status. We find selective expression of BORIS isoform
families in NO, which becomes altered in EOC, primarily by the activation of
BORIS sf1 in EOC. When comparing EOC samples based on methylation status, we
find that BORIS sf1 and sf2 isoform families are selectively activated in
globally hypomethylated tumors. In contrast, CTCF is downregulated in EOC, and
the ratio of BORIS sf1, sf2, and sf6 isoform families as a function of CTCF is
elevated in hypomethylated tumors. Finally, the expression of all BORIS isoform
families was induced to varying extents by epigenetic modulatory drugs in EOC
cell lines, particularly when DNMT and HDAC inhibitors were used in combination. CTCF plays a vital role in chromatin structure and function. CTCF is
ubiquitously expressed and plays diverse roles in gene regulation, imprinting,
insulation, intra/interchromosomal interactions, nuclear compartmentalisation,
and alternative splicing. CTCF has a single paralogue, the testes-specific
CTCF-like gene (CTCFL)/BORIS. CTCF and BORIS can be deregulated in cancer. The
tumour suppressor gene CTCF can be mutated or deleted in cancer, or CTCF DNA
binding can be altered by epigenetic changes. BORIS is aberrantly expressed
frequently in cancer, leading some to propose a pro-tumourigenic role for BORIS.
However, BORIS can inhibit cell proliferation, and is mutated in cancer
similarly to CTCF suggesting BORIS activation in cancer may be due to global
genetic or epigenetic changes typical of maligt transformation. |
What is the application of the ASSET algorithm in C.elegans? | ASSET (Algorithm for the Segmentation and the Standardization of C. elegans Time-lapse recordings) is a robust algorithm for the automated segmentation and standardization of early Caenorhabditis elegans embryos. It gathers quantitative information with subcellular precision in the early Caenorhabditis elegans embryo, which is an attractive model to investigate evolutionarily conserved cellular mechanisms. ASSET automatically detects the eggshell and the cell cortex from DIC time-lapse recordings of live one-cell-stage embryos and can also track subcellular structures using fluorescent time-lapse microscopy. Importantly, ASSET standardizes the data into an absolute coordinate system to allow robust quantitative comparisons between embryos. | |
Are Sidekick proteins members of the immunoglobulin superfamily? | Yes, sidekick are cell adhesion molecules of the immunoglobulin superfamily. | |
What are the pyknons? | Using an unsupervised pattern-discovery method, the human intergenic and intronic regions were processed and all variable-length patterns with identically conserved copies and multiplicities above what is expected by chance were catalogued. Among the millions of discovered patterns, a subset of 127,998 patterns was found, termed pyknons, which have additional nonoverlapping instances in the untranslated and protein-coding regions of 30,675 transcripts from 20,059 human genes. The pyknons arrange combinatorially in the untranslated and coding regions of numerous human genes where they form mosaics. Pyknons might represent a biologically important link between coding and non-coding DNA. | Pyknons are non-random sequence patterns significantly repeated throughout
non-coding genomic DNA that also appear at least once among coding genes. They
are interesting because they portend an unforeseen connection between coding and
non-coding DNA. Pyknons have only been discovered in the human genome, so it is
unknown whether pyknons have wider biological relevance or are simply a
phenomenon of the human genome. To address this, DNA sequence patterns from the
Arabidopsis thaliana genome were detected using a probability-based method. 24
654 statistically significant sequence patterns, 16 to 24 nucleotides long,
repeating 10 or more times in non-coding DNA also appeared in 46% of A. thaliana
protein-coding genes. A. thaliana pyknons exhibit features similar to human
pyknons, including being distinct sequence patterns, having multiple instances
in genes and having remarkable similarity to small RNA sequences with roles in
gene silencing. Chromosomal position mapping revealed that genomic pyknon
density has concordance with siRNA and transposable element positioning density.
Because the A. thaliana and human genomes have approximately the same number of
genes but drastically different amounts of non-coding DNA, these data reveal
that pyknons represent a biologically important link between coding and
non-coding DNA. Because of the association of pyknons with siRNAs and
localization to silenced regions of heterochromatin, we postulate that
RNA-mediated gene silencing leads to the accumulation of gene sequences in
non-coding DNA regions. |
Which deiodinases are best known to be present in brain? | All the 3 deiodinases (Type 1, Type 2 and Type 3 deiodinase) are present in the "brain" but Type 1 deiodinase is only found in neurohypophysis that cannot be actually considered true "brain tissue". | In the present study the hypothesis was tested that
N-bromoacetyl-3,3',5-[125I]triiodothyronine (BrAc[125I]T3) is a useful affinity
label for both type I and type III iodothyronine deiodinases (ID-I and ID-III).
Therefore, the microsomal fractions of various rat tissues were tested for ID-I
and ID-III activities, and microsomal proteins were labeled with BrAc[125I]T3
and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I
activities were found in liver, kidney and thyroid, and high ID-III activities
in brain, in particular fetal brain, and placenta. SDS-PAGE of
BrAc[125I]T3-labeled microsomes showed a prominent radioactive approximately 27
kDa protein (p27) in liver, kidney and thyroid, which was previously identified
as ID-I, and a approximately 32 kDa protein (p32) in brain, in particular fetal
brain, and placenta. A good correlation was found between the affinity labeling
of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents
ID-III or a subunit thereof. After treatment of microsomes with 0.05%
deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by
BrAc[125I]T3, indicating that p32 is a transmembrane protein. Although
3,3',5'-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily
labeled with BrAc[125I]rT3. Labeling of p32 in rat brain microsomes by
BrAc[125I]rT3 was not affected by addition of 100 microM unlabeled thyroxine
(T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and
96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT
250 WORDS) In the brain, thyroid hormone dynamically regulates levels of the short-lived
plasma membrane protein, type II iodothyronine 5'-deiodinase. In cultured
astrocytes, thyroxine modulates deiodinase levels by activating
cytoskeletal-plasma membrane interactions that increase the rate of inactivation
of the enzyme. Here we characterized the effects of these thyroxine-dependent
cytoskeletal interactions upon the route of internalization of the deiodinase by
following the intracellular transit of the affinity-labeled substrate-binding
subunit of the deiodinase (p29). Thyroxine rapidly induced the inactivation of
the deiodinase and initiated the binding of p29 to F-actin. By 40 min, > 75% of
the p29 had been transported to an endosomal pool, which was followed by
dissociation of the F-actin-p29 complex. There was no significant accumulation
of p29 in the dense lysosomes seen in the presence of thyroxine. In the absence
of thyroxine, p29 was internalized and transported to the dense lysosomes at a
rate parallel to the inactivation rate of the deiodinase (t1/2 0.75 and 0.64 h,
respectively) without involvement with the microfilaments. These data
demonstrate that thyroxine targets type II iodothyronine 5'-deiodinase to an
endosomal pool by activating specific protein-F-actin interactions involved in
microfilament-mediated intracellular protein trafficking. Thyroxine dynamically regulates levels of type II iodothyronine 5'-deiodinase
(5'D-II) by modulating enzyme inactivation and targeting the enzyme to different
pathways of internalization. 5'D-II is an approximately 200-kDa multimeric
protein containing a 29-kDa substrate-binding subunit (p29) and an unknown
number of other subunits. In the absence of thyroxine (T4), p29 is slowly
endocytosed and transported to the lysosomes. T4 treatment rapidly activates an
actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In
this study, we have characterized the influence of T4 on the intracellular
trafficking of 5'D-II. We show that T4 accelerates the rate of 5'D-II
inactivation by translocating the enzyme to the interior of the cell and by
sequestering p29 in the endosomal pool without accelerating the rate of
degradation of p29. This dichotomy between the rapid inactivation of catalytic
activity and the much slower degradation of p29 is consistent with the reuse of
p29 in the production of 5'D-II activity. Immunocytochemical analysis with a
specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the
plasma membrane approximately 2 h after enzyme internalization in the presence
of T4, indicating that p29 is recycled. Despite the ability of p29 to be
recycled in the T4-treated cell, 5'D-II catalytic activity requires ongoing
protein synthesis, presumably of another enzyme component(s) or an accessory
enzyme-related protein. In the absence of T4, enzyme inactivation and p29
degradation are temporally linked, and pulse affinity-labeled p29 is
internalized and sequestered in discrete intracellular pools. These data suggest
that T4 regulates fundamental processes involved with the turnover of integral
membrane proteins and participates in regulating the inter-relationships between
the degradation, recycling, and synthetic pathways. Intracellular generation of triiodothyronine (T3) from thyroxine (T4) by type 2
deiodinase (D2) in the mammalian brain, plays a key role in thyroid hormone
action. The presence of D2 in rat astrocytes suggests the importance of glial
cells in the regulation of intracellular T3 levels in the rat central nervous
system (CNS). To analyze further the factors that regulate D2 activity in the
CNS, we investigated the effects of nicotine and of mecamylamine, which inhibits
the binding of nicotine with nicotinic acetylcholine receptors, on D2 activity
in cultured mixed glial cells of the rat brain. We incubated cultured mixed
glial cells obtained from neonatal Wistar rats in the presence of 10 mM
dithiothreitol, 2 nM [125I] reverse T3 and 1 mM 6-N-propyl-2-thiouracil for 2 h
at 37 degrees C, and the released 125I- was counted in a gamma counter. D2
activity of cultured cells was dependent on the temperature and the amount of
protein. The basal D2 activity of rat mixed glial cells was 1.9 +/- 0.2 fmol of
I- released/mg protein/h (mean +/- SEM). The addition of 10(-11), 2 x 10(-11),
10(-10), and 10(-9) M nicotine significantly increased D2 activity to
approximately 2.2-, 2.4, 3.5- and 2.9-fold the basal level, respectively. D2
activity stimulated by 10(-8) M nicotine (2.5-fold) reached a peak after 9 h
incubation. The stimulatory effect of nicotine was completely blocked by 10(-6)
M mecamylamine. In conclusion, nicotine increases D2 activity probably via
nicotinic acetylcholine receptors, and may influence brain function, at least in
part, by affecting thyroid hormone metabolism. Because iodothyronine deiodinases play a crucial role in the regulation of the
available intracellular T(3) concentration, it is important to determine their
cellular localization. In brain, the presence of type III iodothyronine
deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels.
Until now, no cellular localization pattern of the D3 protein was reported in
chicken brain. In this study polyclonal antisera were produced against specific
peptides corresponding to the D3 amino acid sequence. Their use in
immunocytochemistry led to the localization of D3 in the Purkinje cells of the
chicken cerebellum. Both preimmune serum as well as the primary antiserum
exhausted with the peptide itself were used as negative controls. Extracts of
chick cerebellum and liver were made in the presence of Triton X-100 to
solubilize the membrane-bound deiodinases. Using these extracts in Western blot
analysis, a band of the expected molecular weight ( approximately 30 kDa) could
be detected in both tissues. Using a full-length (32)P-labeled type III
deiodinase cRNA probe, we identified a single mRNA species in the cerebellum
that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR,
followed by subcloning and sequence analysis, confirmed the expression of D3
mRNA in the chicken cerebellum. In this study we provide the first evidence of
the presence of the D3 protein in a neuronal cell type, namely Purkinje cells,
by means of immunocytochemical staining. We were able to detect a protein
fragment corresponding to the expected molecular mass (30 kDa) for type III
deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot
analysis confirmed the presence of D3 mRNA in the cerebellum. OBJECTIVE: The determits of response to antidepressant treatment in major
depression are unknown at present. The aim of the present study was to establish
whether response is predicted by Hypothalamus-Pituitary-Thyroid (HPT) axis
parameters or by a recently discovered polymorphism in the enzyme type II
deiodinase (DII), which catalyzes the production of T3 in the brain.
DESIGN: We analyzed prediction of response to paroxetine treatment by
calculating response rates per tertile of HPT-axis parameters and per DII
genotype.
METHODS: Ninety-eight outpatients with major depression (DSM-IV) were included.
Serum concentrations of TSH, FT4 and delta TSH in a DEX/CRH-TRH test were
measured. In addition, the presence of a polymorphism in the DII sequence
(Thr92Ala) was determined.
RESULTS: The overall treatment response was 48 of 98 patients (49%). After
exclusion of patients with subclinical hypothyroidism and/or TPO antibodies (n =
16), higher serum TSH significantly predicted response (response rate per
tertile from low to high TSH: 36%, 42%, and 67%). Heterozygous patients for the
DII polymorphism (44%) had slightly lower serum TSH (P = 0.03) as compared to
patients with the wild-type DII (47%). The polymorphism was unrelated to
treatment response.
CONCLUSION: Higher serum TSH was associated with response to paroxetine in
patients with major depression. Organic anion-transporting polypeptide 1c1 (Oatp1c1) (also known as Slco1c1 and
Oatp14) belongs to the family of Oatp and has been shown to facilitate the
transport of T(4). In the rodent brain, Oatp1c1 is highly enriched in capillary
endothelial cells and choroid plexus structures where it may mediate the entry
of T(4) into the central nervous system. Here, we describe the generation and
first analysis of Oatp1c1-deficient mice. Oatp1c1 knockout (KO) mice were born
with the expected frequency, were not growth retarded, and developed without any
overt neurological abnormalities. Serum T(3) and T(4) concentrations as well as
renal and hepatic deiodinase type 1 expression levels were indistinguishable
between Oatp1c1 KO mice and control animals. Hypothalamic TRH and pituitary TSH
mRNA levels were not affected, but brain T(4) and T(3) content was decreased in
Oatp1c1-deficient animals. Moreover, increased type 2 and decreased type 3
deiodinase activities indicate a mild hypothyroid situation in the brain of
Oatp1c1 KO mice. Consequently, mRNA expression levels of gene products
positively regulated by T(3) in the brain were down-regulated. This central
nervous system-specific hypothyroidism is presumably caused by an impaired
passage of T(4) across the blood-brain barrier and indicates a unique function
of Oatp1c1 in facilitating T(4) transport despite the presence of other thyroid
hormone transporters such as Mct8. Hypothalamic neurosecretory systems are fundamental regulatory circuits
influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated
uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed
inactivation represent limiting regulatory factors of neuronal T3 availability.
In the present study we addressed the localization and subcellular distribution
of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their
axons. Intense D3-immunoreactivity was observed in axon varicosities in the
external zone of the rat median eminence and the neurohaemal zone of the human
infundibulum containing axon terminals of hypophysiotropic parvocellular
neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in
hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected
the active center containing C-terminus of D3 at the outer surface of these
organelles. Double-labeling immunofluorescent confocal microscopy revealed that
D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority
of TRH axons, while absent from somatostatin-containing neurons.
Bimolecular-Fluorescence-Complementation identified D3 homodimers, a
prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore,
T3-inducible D3 catalytic activity was detected in the rat median eminence.
Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the
presence of MCT8 on the surface of the vast majority of all types of
hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the
axon terminals in the neurohaemal zone of the human infundibulum. The unexpected
role of hypophysiotropic axons in fine-tuned regulation of T3 availability in
these cells via MCT8-mediated transport and D3-catalyzed inactivation may
represent a novel regulatory core mechanism for metabolism, growth, stress and
reproduction in rodents and humans. |
What are the computational methods for the prediction of beta-barrel transmembrane proteins? | Computational tools have been developed for beta-barrel transmembrane protein discrimination, topology prediction and prediction of their structural features.
Initial methods developed for the prediction of the transmembrane beta strands were based on hydrophobicity analysis, using sliding windows along the sequence, in order to capture the alternating patterns of hydrophobic-hydrophilic residues of the transmembrane strands, or using generalized secondary structure prediction methods. Other approaches included the construction of special empirical rules using amino-acid propensities and prior knowledge of the structural nature of the proteins, and the development of Neural Network-based predictors to predict the location of alpha-carbon atoms with respect to the membrane. During the last few years, other more refined methods, appeared, including: Neural Networks, Hidden Markov Models, Support Vector Machines, k-Nearest Neighbors, Radial Basis Functions, Bayesian Networks, Genetic Algorithms, Mahalanobis Discriminant Functions, Cellular Automata, N-to-1 Extreme Learning Machines. Hidden Markov Model-based methods are among the most successful in topology prediction, being able to capture the unique architecture of beta-barrel transmembrane proteins. Consensus methods, as well as pipelines of several related tools (e.g. subcellular localization prediciton, alpha-helical transmembrane protein prediction, signal-peptide/lipoprotein prediction) have also used for discriminating beta-barrel transmembrane proteins. Recently, a number of methods for predicting more detailed structural features (e.g. surface accessibility, residue contacts, even detailed atomic 3D models) tailored to beta-barrel transmembrane proteins have been developed, based on knowledge-based potential functions, graph theoretic models, physical models and multi-tape S-attribute grammars. Methods/tools falling in the aforementioned classes are (listed in alphabetical order): BBF (beta-barrel finder), BETAWARE, BOCTOPUS, BOMP, BTMX (Beta barrel TransMembrane eXposure), HHomp, HMM-B2TMR, OMBBpred, PROFtmb, PRED-TMBB, TMB-Hunt, TBBPred, TMBETAPRED-RBF, TMBHMM, TransFold, TMBpro, TMBKNN, Wimley | In order to propose a reliable model for Brucella porin topology, several
structure prediction methods were evaluated in their ability to predict porin
topology. Four porins of known structure were selected as test-cases and their
secondary structure delineated. The specificity and sensitivity of 11 methods
were separately evaluated. Our critical assessment shows that some secondary
structure prediction methods (PHD, Dsc, Sopma) originally designed to predict
globular protein structure are useful on porin topology prediction. The overall
best prediction is obtained by combining these three "generalist" methods with a
transmembrane beta strand prediction technique. This "consensus" method was
applied to Brucella porins Omp2b and Omp2a, sharing no sequence homology with
any other porin. The predicted topology is a 16-stranded antiparallel beta
barrel with Omp2a showing a higher number of negatively charged residue in the
exposed loops than Omp2b. Experiments are in progress to validate the proposed
topology and the functional hypotheses. The ability of the proposed consensus
method to predict topology of complex outer membrane protein is briefly
discussed. A method based on neural networks is trained and tested on a nonredundant set of
beta-barrel membrane proteins known at atomic resolution with a jackknife
procedure. The method predicts the topography of transmembrane beta strands with
residue accuracy as high as 78% when evolutionary information is used as input
to the network. Of the transmembrane beta-strands included in the training set,
93% are correctly assigned. The predictor includes an algorithm of model
optimization, based on dynamic programming, that correctly models eight out of
the 11 proteins present in the training/testing set. In addition, protein
topology is assigned on the basis of the location of the longest loops in the
models. We propose this as a general method to fill the gap of the prediction of
beta-barrel membrane proteins. There is preliminary experimental evidence indicating that the major
outer-membrane protein (MOMP) of Chlamydia is a porin. We tested this hypothesis
for the MOMP of the mouse pneumonitis serovar of Chlamydia trachomatis using two
secondary structure prediction methods. First, an algorithm that calculates the
mean hydrophobicity of one side of putative beta-strands predicted the positions
of 16 transmembrane segments, a structure common to known porins. Second, outer
loops typical of porins were assigned using an artificial neural network trained
to predict the topology of bacterial outer-membrane proteins with a predomice
of beta-strands. A topology model based on these results locates the four
variable domains (VDs) of the MOMP on the outer loops and the five constant
domains on beta-strands and the periplasmic turns. This model is consistent with
genetic analysis and immunological and biochemical data that indicate the VDs
are surface exposed. Furthermore, it shows significant homology with the
consensus porin model of the program FORESST, which contrasts a proposed
secondary structure against a data set of 349 proteins of known structure.
Analysis of the MOMP of other chlamydial species corroborated our predicted
model. Many outer membrane proteins (OMPs) in Gram-negative bacteria possess known
beta-barrel three-dimensional (3D) structures. These proteins, including
channel-forming transmembrane porins, are diverse in sequence but exhibit common
structural features. We here report computational analyses of six outer membrane
proteins of known 3D structures with respect to (1) secondary structure, (2)
hydropathy, and (3) amphipathicity. Using these characteristics, as well as the
presence of an N-terminal targeting sequence, a program was developed allowing
prediction of integral membrane beta-barrel proteins encoded within any
completely sequenced prokaryotic genome. This program, termed the beta-barrel
finder (BBF) program, was used to analyze the proteins encoded within the
Escherichia coli genome. Out of 4290 sequences examined, 118 (2.8%) were
retrieved. Of these, almost all known outer membrane proteins with established
beta-barrel structures as well as many probable outer membrane proteins were
identified. This program should be useful for predicting the occurrence of outer
membrane proteins in bacteria with completely sequenced genomes. A novel method is developed to model and predict the transmembrane regions of
beta-barrel membrane proteins. It is based on a Hidden Markov model (HMM) with
architecture obeying those proteins' construction principles. The HMM is trained
and tested on a non-redundant set of 11 beta-barrel membrane proteins known to
date at atomic resolution with a jack-knife procedure. As a result, the method
correctly locates 97% of 172 transmembrane beta-strands. Out of the 11 proteins,
the barrel size for ten proteins and the overall topology for seven proteins are
correctly predicted. Additionally, it successfully assigns the entire topology
for two new beta-barrel membrane proteins that have no significant sequence
homology to the 11 proteins. Predicted topology for two candidates for
beta-barrel structure of the outer mitochondrial membrane is also presented in
the paper. Very few methods address the problem of predicting beta-barrel membrane proteins
directly from sequence. One reason is that only very few high-resolution
structures for transmembrane beta-barrel (TMB) proteins have been determined
thus far. Here we introduced the design, statistics and results of a novel
profile-based hidden Markov model for the prediction and discrimination of TMBs.
The method carefully attempts to avoid over-fitting the sparse experimental
data. While our model training and scoring procedures were very similar to a
recently published work, the architecture and structure-based labelling were
significantly different. In particular, we introduced a new definition of beta-
hairpin motifs, explicit state modelling of transmembrane strands, and a
log-odds whole-protein discrimination score. The resulting method reached an
overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high
as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45%
coverage at 100% accuracy). This high precision enabled the application to 72
entirely sequenced Gram-negative bacteria. We found over 164 previously
uncharacterized TMB proteins at high confidence. Database searches did not
implicate any of these proteins with membranes. We challenge that the vast
majority of our 164 predictions will eventually be verified experimentally. All
proteome predictions and the PROFtmb prediction method are available at
http://www.rostlab.org/ services/PROFtmb/. This article describes a method developed for predicting transmembrane
beta-barrel regions in membrane proteins using machine learning techniques:
artificial neural network (ANN) and support vector machine (SVM). The ANN used
in this study is a feed-forward neural network with a standard back-propagation
training algorithm. The accuracy of the ANN-based method improved significantly,
from 70.4% to 80.5%, when evolutionary information was added to a single
sequence as a multiple sequence alignment obtained from PSI-BLAST. We have also
developed an SVM-based method using a primary sequence as input and achieved an
accuracy of 77.4%. The SVM model was modified by adding 36 physicochemical
parameters to the amino acid sequence information. Finally, ANN- and SVM-based
methods were combined to utilize the full potential of both techniques. The
accuracy and Matthews correlation coefficient (MCC) value of SVM, ANN, and
combined method are 78.5%, 80.5%, and 81.8%, and 0.55, 0.63, and 0.64,
respectively. These methods were trained and tested on a nonredundant data set
of 16 proteins, and performance was evaluated using "leave one out
cross-validation" (LOOCV). Based on this study, we have developed a Web server,
TBBPred, for predicting transmembrane beta-barrel regions in proteins (available
at http://www.imtech.res.in/raghava/tbbpred). This work describes the development of a program that predicts whether or not a
polypeptide sequence from a Gram-negative bacterium is an integral beta-barrel
outer membrane protein. The program, called the beta-barrel Outer Membrane
protein Predictor (BOMP), is based on two separate components to recognize
integral beta-barrel proteins. The first component is a C-terminal pattern
typical of many integral beta-barrel proteins. The second component calculates
an integral beta-barrel score of the sequence based on the extent to which the
sequence contains stretches of amino acids typical of transmembrane
beta-strands. The precision of the predictions was found to be 80% with a recall
of 88% when tested on the proteins with SwissProt annotated subcellular
localization in Escherichia coli K 12 (788 sequences) and Salmonella typhimurium
(366 sequences). When tested on the predicted proteome of E.coli, BOMP found 103
of a total of 4346 polypeptide sequences to be possible integral beta-barrel
proteins. Of these, 36 were found by BLAST to lack similarity (E-value score <
1e-10) to proteins with annotated subcellular localization in SwissProt. BOMP
predicted the content of integral beta-barrels per predicted proteome of 10
different bacteria to range from 1.8 to 3%. BOMP is available at
http://www.bioinfo.no/tools/bomp. The beta-barrel outer membrane proteins constitute one of the two known
structural classes of membrane proteins. Whereas there are several different
web-based predictors for alpha-helical membrane proteins, currently there is no
freely available prediction method for beta-barrel membrane proteins, at least
with an acceptable level of accuracy. We present here a web server (PRED-TMBB,
http://bioinformatics.biol.uoa.gr/PRED-TMBB) which is capable of predicting the
transmembrane strands and the topology of beta-barrel outer membrane proteins of
Gram-negative bacteria. The method is based on a Hidden Markov Model, trained
according to the Conditional Maximum Likelihood criterion. The model was
retrained and the training set now includes 16 non-homologous outer membrane
proteins with structures known at atomic resolution. The user may submit one
sequence at a time and has the option of choosing between three different
decoding methods. The server reports the predicted topology of a given protein,
a score indicating the probability of the protein being an outer membrane
beta-barrel protein, posterior probabilities for the transmembrane strand
prediction and a graphical representation of the assumed position of the
transmembrane strands with respect to the lipid bilayer. BACKGROUND: Prediction of the transmembrane strands and topology of beta-barrel
outer membrane proteins is of interest in current bioinformatics research.
Several methods have been applied so far for this task, utilizing different
algorithmic techniques and a number of freely available predictors exist. The
methods can be grossly divided to those based on Hidden Markov Models (HMMs), on
Neural Networks (NNs) and on Support Vector Machines (SVMs). In this work, we
compare the different available methods for topology prediction of beta-barrel
outer membrane proteins. We evaluate their performance on a non-redundant
dataset of 20 beta-barrel outer membrane proteins of gram-negative bacteria,
with structures known at atomic resolution. Also, we describe, for the first
time, an effective way to combine the individual predictors, at will, to a
single consensus prediction method.
RESULTS: We assess the statistical significance of the performance of each
prediction scheme and conclude that Hidden Markov Model based methods,
HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according
to either the per-residue accuracy, the segments overlap measure (SOV) or the
total number of proteins with correctly predicted topologies in the test set.
Furthermore, we show that the available predictors perform better when only
transmembrane beta-barrel domains are used for prediction, rather than the
precursor full-length sequences, even though the HMM-based predictors are not
influenced significantly. The consensus prediction method performs significantly
better than each individual available predictor, since it increases the accuracy
up to 4% regarding SOV and up to 15% in correctly predicted topologies.
CONCLUSIONS: The consensus prediction method described in this work, optimizes
the predicted topology with a dynamic programming algorithm and is implemented
in a web-based application freely available to non-commercial users at
http://bioinformatics.biol.uoa.gr/ConBBPRED. BACKGROUND: Beta-barrel transmembrane (bbtm) proteins are a functionally
important and diverse group of proteins expressed in the outer membranes of
bacteria (both gram negative and acid fast gram positive), mitochondria and
chloroplasts. Despite recent publications describing reasonable levels of
accuracy for discriminating between bbtm proteins and other proteins, screening
of entire genomes remains troublesome as these molecules only constitute a small
fraction of the sequences screened. Therefore, novel methods are still required
capable of detecting new families of bbtm protein in diverse genomes.
RESULTS: We present TMB-Hunt, a program that uses a k-Nearest Neighbour (k-NN)
algorithm to discriminate between bbtm and non-bbtm proteins on the basis of
their amino acid composition. By including differentially weighted amino acids,
evolutionary information and by calibrating the scoring, an accuracy of 92.5%
was achieved, with 91% sensitivity and 93.8% positive predictive value (PPV),
using a rigorous cross-validation procedure. A major advantage of this approach
is that because it does not rely on beta-strand detection, it does not require
resolved structures and thus larger, more representative, training sets could be
used. It is therefore believed that this approach will be invaluable in
complementing other, physicochemical and homology based methods. This was
demonstrated by the correct reassignment of a number of proteins which other
predictors failed to classify. We have used the algorithm to screen several
genomes and have discussed our findings.
CONCLUSION: TMB-Hunt achieves a prediction accuracy level better than other
approaches published to date. Results were significantly enhanced by use of
evolutionary information and a system for calibrating k-NN scoring. Because the
program uses a distinct approach to that of other discriminators and thus
suffers different liabilities, we believe it will make a significant
contribution to the development of a consensus approach for bbtm protein
detection. BACKGROUND: Hidden Markov Models (HMMs) have been extensively used in
computational molecular biology, for modelling protein and nucleic acid
sequences. In many applications, such as transmembrane protein topology
prediction, the incorporation of limited amount of information regarding the
topology, arising from biochemical experiments, has been proved a very useful
strategy that increased remarkably the performance of even the top-scoring
methods. However, no clear and formal explanation of the algorithms that retains
the probabilistic interpretation of the models has been presented so far in the
literature.
RESULTS: We present here, a simple method that allows incorporation of prior
topological information concerning the sequences at hand, while at the same time
the HMMs retain their full probabilistic interpretation in terms of conditional
probabilities. We present modifications to the standard Forward and Backward
algorithms of HMMs and we also show explicitly, how reliable predictions may
arise by these modifications, using all the algorithms currently available for
decoding HMMs. A similar procedure may be used in the training procedure, aiming
at optimizing the labels of the HMM's classes, especially in cases such as
transmembrane proteins where the labels of the membrane-spanning segments are
inherently misplaced. We present an application of this approach developing a
method to predict the transmembrane regions of alpha-helical membrane proteins,
trained on crystallographically solved data. We show that this method compares
well against already established algorithms presented in the literature, and it
is extremely useful in practical applications.
CONCLUSION: The algorithms presented here, are easily implemented in any kind of
a Hidden Markov Model, whereas the prediction method (HMM-TM) is freely
available for academic users at http://bioinformatics.biol.uoa.gr/HMM-TM,
offering the most advanced decoding options currently available. In this study, we propose a novel method to predict the solvent accessible
surface areas of transmembrane residues. For both transmembrane alpha-helix and
beta-barrel residues, the correlation coefficients between the predicted and
observed accessible surface areas are around 0.65. On the basis of predicted
accessible surface areas, residues exposed to the lipid environment or buried
inside a protein can be identified by using certain cutoff thresholds. We have
extensively examined our approach based on different definitions of accessible
surface areas and a variety of sets of control parameters. Given that
experimentally determining the structures of membrane proteins is very difficult
and membrane proteins are actually abundant in nature, our approach is useful
for theoretically modeling membrane protein tertiary structures, particularly
for modeling the assembly of transmembrane domains. This approach can be used to
annotate the membrane proteins in proteomes to provide extra structural and
functional information. Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of
Gram-negative bacteria, mitochondria and chloroplasts. The cellular location and
functional diversity of beta-barrel outer membrane proteins makes them an
important protein class. At the present time, very few non-homologous TMB
structures have been determined by X-ray diffraction because of the experimental
difficulty encountered in crystallizing transmembrane (TM) proteins. The
transFold web server uses pairwise inter-strand residue statistical potentials
derived from globular (non-outer-membrane) proteins to predict the
supersecondary structure of TMB. Unlike all previous approaches, transFold does
not use machine learning methods such as hidden Markov models or neural
networks; instead, transFold employs multi-tape S-attribute grammars to describe
all potential conformations, and then applies dynamic programming to determine
the global minimum energy supersecondary structure. The transFold web server not
only predicts secondary structure and TMB topology, but is the only method which
additionally predicts the side-chain orientation of transmembrane beta-strand
residues, inter-strand residue contacts and TM beta-strand inclination with
respect to the membrane. The program transFold currently outperforms all other
methods for accuracy of beta-barrel structure prediction. Available at
http://bioinformatics.bc.edu/clotelab/transFold. Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of
Gram-negative bacteria, mitochondria, and chloroplasts. The cellular location
and functional diversity of beta-barrel outer membrane proteins (omps) makes
them an important protein class. At the present time, very few nonhomologous TMB
structures have been determined by X-ray diffraction because of the experimental
difficulty encountered in crystallizing transmembrane proteins. A novel method
using pairwise interstrand residue statistical potentials derived from globular
(nonouter membrane) proteins is introduced to predict the supersecondary
structure of transmembrane beta-barrel proteins. The algorithm transFold employs
a generalized hidden Markov model (i.e., multitape S-attribute grammar) to
describe potential beta-barrel supersecondary structures and then computes by
dynamic programming the minimum free energy beta-barrel structure. Hence, the
approach can be viewed as a "wrapping" component that may capture folding
processes with an initiation stage followed by progressive interaction of the
sequence with the already-formed motifs. This approach differs significantly
from others, which use traditional machine learning to solve this problem,
because it does not require a training phase on known TMB structures and is the
first to explicitly capture and predict long-range interactions. TransFold
outperforms previous programs for predicting TMBs on smaller (<or=200 residues)
proteins and matches their performance for straightforward recognition of longer
proteins. An exception is for multimeric porins where the algorithm does perform
well when an important functional motif in loops is initially identified. We
verify our simulations of the folding process by comparing them with
experimental data on the functional folding of TMBs. A Web server running
transFold is available and outputs contact predictions and locations for
sequences predicted to form TMBs. We have developed the database, TMBETA-GENOME, for annotated beta-barrel
membrane proteins in genomic sequences using statistical methods and machine
learning algorithms. The statistical methods are based on amino acid
composition, reside pair preference and motifs. In machine learning techniques,
the combination of amino acid and dipeptide compositions has been used as main
attributes. In addition, annotations have been made using the criterion based on
the identification of beta-barrel membrane proteins and exclusion of globular
and transmembrane helical proteins. A web interface has been developed for
identifying the annotated beta-barrel membrane proteins in all known genomes.
The users have the feasibility of selecting the genome from the three kingdoms
of life, archaea, bacteria and eukaryote, and five different methods. Further,
the statistics for all genomes have been provided along with the links to
different algorithms and related databases. It is freely available at
http://tmbeta-genome.cbrc.jp/annotation/. Transmembrane (TM) proteins represent about 20-30% of the protein sequences in
higher eukaryotes, playing important roles across a range of cellular functions.
Moreover, knowledge about topology of these proteins often provides crucial
hints toward their function. Due to the difficulties in experimental structure
determinations of TM protein, theoretical prediction methods are highly
preferred in identifying the topology of newly found ones according to their
primary sequences, useful in both basic research and drug discovery. In this
paper, based on the concept of pseudo amino acid composition (PseAA) that can
incorporate sequence-order information of a protein sequence so as to remarkably
enhance the power of discrete models (Chou, K. C., Proteins: Structure,
Function, and Genetics, 2001, 43: 246-255), cellular automata and Lempel-Ziv
complexity are introduced to predict the TM regions of integral membrane
proteins including both alpha-helical and beta-barrel membrane proteins,
validated by jackknife test. The result thus obtained is quite promising, which
indicates that the current approach might be a quite potential high throughput
tool in the post-genomic era. The source code and dataset are available for
academic users at [email protected]. Accurate protein structure prediction remains an active objective of research in
bioinformatics. Membrane proteins comprise approximately 20% of most genomes.
They are, however, poorly tractable targets of experimental structure
determination. Their analysis using bioinformatics thus makes an important
contribution to their on-going study. Using a method based on Bayesian Networks,
which provides a flexible and powerful framework for statistical inference, we
have addressed the alignment-free discrimination of membrane from non-membrane
proteins. The method successfully identifies prokaryotic and eukaryotic
alpha-helical membrane proteins at 94.4% accuracy, beta-barrel proteins at 72.4%
accuracy, and distinguishes assorted non-membranous proteins with 85.9%
accuracy. The method here is an important potential advance in the computational
analysis of membrane protein structure. It represents a useful tool for the
characterisation of membrane proteins with a wide variety of potential
applications. Membrane proteins, which constitute approximately 20% of most genomes, form two
main classes: alpha helical and beta barrel transmembrane proteins. Using
methods based on Bayesian Networks, a powerful approach for statistical
inference, we have sought to address beta-barrel topology prediction. The
beta-barrel topology predictor reports individual strand accuracies of 88.6%.
The method outlined here represents a potentially important advance in the
computational determination of membrane protein topology. We have developed a novel approach for dissecting transmembrane beta-barrel
proteins (TMBs) in genomic sequences. The features include (i) the
identification of TMBs using the preference of residue pairs in globular,
transmembrane helical (TMH) and TMBs, (ii) elimination of globular/TMH proteins
that show sequence identity of more than 70% for the coverage of 80% residues
with known structures, (iii) elimination of globular/TMH proteins that have
sequence identity of more than 60% with known sequences in SWISS-PROT, and (iv)
exclusion of TMH proteins using SOSUI, a prediction system for TMH proteins. Our
approach picked up 7% TMBs in all the considered genomes. The comparison between
the identified TMBs in E. coli genome and available experimental data
demonstrated that the new approach could correctly identify all the 11 known
TMBs, whose crystal structures are available. Further, it revealed the presence
of 19 TMBs, homology with known structures, 60 TMBs similar to well annotated
sequences, and 54 TMBs that have high sequence similarity with Escherichia coli
beta-barrel proteins deposited in Transport Classification Database (TCDB).
Interestingly, the present approach identified TMBs from all 15 families in
TCDB. In human genome, the occurrence of TMBs varies from 0 to 3% in different
chromosomes. We suggest that our approach could lead to a step forward in the
advancement of structural and functional genomics. MOTIVATION: Transmembrane beta-barrel (TMB) proteins are embedded in the outer
membranes of mitochondria, Gram-negative bacteria and chloroplasts. These
proteins perform critical functions, including active ion-transport and passive
nutrient intake. Therefore, there is a need for accurate prediction of secondary
and tertiary structure of TMB proteins. Traditional homology modeling methods,
however, fail on most TMB proteins since very few non-homologous TMB structures
have been determined. Yet, because TMB structures conform to specific
construction rules that restrict the conformational space drastically, it should
be possible for methods that do not depend on target-template homology to be
applied successfully.
RESULTS: We develop a suite (TMBpro) of specialized predictors for predicting
secondary structure (TMBpro-SS), beta-contacts (TMBpro-CON) and tertiary
structure (TMBpro-3D) of transmembrane beta-barrel proteins. We compare our
results to the recent state-of-the-art predictors transFold and PRED-TMBB using
their respective benchmark datasets, and leave-one-out cross-validation. Using
the transFold dataset TMBpro predicts secondary structure with per-residue
accuracy (Q(2)) of 77.8%, a correlation coefficient of 0.54, and TMBpro predicts
beta-contacts with precision of 0.65 and recall of 0.67. Using the PRED-TMBB
dataset, TMBpro predicts secondary structure with Q(2) of 88.3% and a
correlation coefficient of 0.75. All of these performance results exceed
previously published results by 4% or more. Working with the PRED-TMBB dataset,
TMBpro predicts the tertiary structure of transmembrane segments with RMSD <6.0
A for 9 of 14 proteins. For 6 of 14 predictions, the RMSD is <5.0 A, with a
GDT_TS score greater than 60.0.
AVAILABILITY: http://www.igb.uci.edu/servers/psss.html. The outer membrane proteins (OMPs) are beta-barrel membrane proteins that
performed lots of biology functions. The discriminating OMPs from other non-OMPs
is a very important task for understanding some biochemical process. In this
study, a method that combines increment of diversity with modified Mahalanobis
Discrimit, called IDQD, is presented to predict 208 OMPs, 206 transmembrane
helical proteins (TMHPs) and 673 globular proteins (GPs) by using Chou's pseudo
amino acid compositions as parameters. The overall accuracy of jackknife
cross-validation is 93.2% and 96.1%, respectively, for three datasets (OMPs,
TMHPs and GPs) and two datasets (OMPs and non-OMPs). These predicted results
suggest that the method can be effectively applied to discriminate OMPs, TMHPs
and GPs. And it also indicates that the pseudo amino acid composition can better
reflect the core feature of membrane proteins than the classical amino acid
composition. We identify and describe a set of tools readily available for integral membrane
protein prediction. These tools address two problems: finding potential
transmembrane proteins in a pool of new sequences, and identifying their
transmembrane regions. All methods involve comparing the query protein against
one or more target models. In the simplest of these, the target "model" is
another protein sequence, while the more elaborate methods group together the
entire set of t ansmembrane helical or transmembrane beta-barrel proteins. In
general, prediction accuracy either in identifying new integral membrane
proteins or transmembrane regions of known integral membrane proteins depends
strongly on how closely the query fits the model. Because of this, the best
approach is an opportunistic one: submit the protein of interest to all methods
and choose the results with the highest confidence scores. Prediction of membrane spanning segments in beta-barrel outer membrane proteins
(OMP) and their topology is an important problem in structural and functional
genomics. In this work, we propose a method based on radial basis networks for
predicting the number of beta-strands in OMPs and identifying their membrane
spanning segments. Our method showed a leave-one-out cross validation accuracy
of 96% in a set of 28 OMPs, which have the range of 8-22 beta-strand segments.
The beta-strand segments in OMPs and the residues in membrane spanning segments
are correctly predicted with the accuracy of 96% and 87%, respectively. We have
developed a web server, TMBETAPRED-RBF for predicting the transmembrane
beta-strands from amino acid sequence and it is available at
http://rbf.bioinfo.tw/~sachen/tmrbf.html. We suggest that our method could be an
effective tool for predicting the membrane spanning regions and topology of
beta-barrel membrane proteins. Outer membrane proteins (OMPs) are the transmembrane proteins found in the outer
membranes of Gram-negative bacteria, mitochondria and plastids. Most prediction
methods have focused on analogous features, such as alternating hydrophobicity
patterns. Here, we start from the observation that almost all beta-barrel OMPs
are related by common ancestry. We identify proteins as OMPs by detecting their
homologous relationships to known OMPs using sequence similarity. Given an input
sequence, HHomp builds a profile hidden Markov model (HMM) and compares it with
an OMP database by pairwise HMM comparison, integrating OMP predictions by
PROFtmb. A crucial ingredient is the OMP database, which contains profile HMMs
for over 20,000 putative OMP sequences. These were collected with the
exhaustive, transitive homology detection method HHsenser, starting from 23
representative OMPs in the PDB database. In a benchmark on TransportDB, HHomp
detects 63.5% of the true positives before including the first false positive.
This is 70% more than PROFtmb, four times more than BOMP and 10 times more than
TMB-Hunt. In Escherichia coli, HHomp identifies 57 out of 59 known OMPs and
correctly assigns them to their functional subgroups. HHomp can be accessed at
http://toolkit.tuebingen.mpg.de/hhomp. BACKGROUND: Alpha-helical transmembrane (TM) proteins are involved in a wide
range of important biological processes such as cell signaling, transport of
membrane-impermeable molecules, cell-cell communication, cell recognition and
cell adhesion. Many are also prime drug targets, and it has been estimated that
more than half of all drugs currently on the market target membrane proteins.
However, due to the experimental difficulties involved in obtaining high quality
crystals, this class of protein is severely under-represented in structural
databases. In the absence of structural data, sequence-based prediction methods
allow TM protein topology to be investigated.
RESULTS: We present a support vector machine-based (SVM) TM protein topology
predictor that integrates both signal peptide and re-entrant helix prediction,
benchmarked with full cross-validation on a novel data set of 131 sequences with
known crystal structures. The method achieves topology prediction accuracy of
89%, while signal peptides and re-entrant helices are predicted with 93% and 44%
accuracy respectively. An additional SVM trained to discriminate between
globular and TM proteins detected zero false positives, with a low false
negative rate of 0.4%. We present the results of applying these tools to a
number of complete genomes. Source code, data sets and a web server are freely
available from http://bioinf.cs.ucl.ac.uk/psipred/.
CONCLUSION: The high accuracy of TM topology prediction which includes detection
of both signal peptides and re-entrant helices, combined with the ability to
effectively discriminate between TM and globular proteins, make this method
ideally suited to whole genome annotation of alpha-helical transmembrane
proteins. BACKGROUND: Hidden Markov models (HMMs) have been extensively used in
computational molecular biology, for modelling protein and nucleic acid
sequences. The design of the model architecture and the algorithms for parameter
estimation and decoding are extremely important for improve the performance of
HMM. In topology prediction of transmembrane beta-barrels proteins (TMBs), the
Baum-Welch algorithm is widely adapted for HMM training but usually leads to a
sub-optimal model in practice. In addition, all the existing HMM-based
predictors are only designed to model the transmembrane segment without a
submodel to model the signal peptide (SP) for full-length sequences. It is not
convenient for users to investigate the structures of full-length TMB sequences.
RESULTS: We present here, an HMM that combine a transmembrane barrel submodel
and an SP submodel for both topology and SP predictions. A new genetic algorithm
(GA) is presented here to training the model, at the same time the
Posterior-Viterbi algorithm is adopted for decoding. A dataset including 33 TMBs
that is the most so far in literature are collected for model training and
testing. Results of self-consistency and jackknife tests shows the GA has better
global performance than the Baum-Welch algorithm. Results of jackknife tests
show that this method performs better than all well known existing methods for
topology predictions. Furthermore, it provides a function to predict SP in
full-length TMBs sequences with fairish accuracy.
CONCLUSION: We show that our combined HMM-based method is a better choice for
TMB topology prediction, which implements topology predictions with higher
accuracy and additional SP predictions for full-length TMB sequences. Transmembrane beta barrel (TMB) proteins are found in the outer membranes of
bacteria, mitochondria and chloroplasts. TMBs are involved in a variety of
functions such as mediating flux of metabolites and active transport of
siderophores, enzymes and structural proteins, and in the translocation across
or insertion into membranes. We present here TMBHMM, a computational method
based on a hidden Markov model for predicting the structural topology of
putative TMBs from sequence. In addition to predicting transmembrane strands,
TMBHMM also predicts the exposure status (i.e., exposed to the membrane or
hidden in the protein structure) of the residues in the transmembrane region,
which is a novel feature of the TMBHMM method. Furthermore, TMBHMM can also
predict the membrane residues that are not part of beta barrel forming strands.
The training of the TMBHMM was performed on a non-redundant data set of 19 TMBs.
The self-consistency test yielded Q(2) accuracy of 0.87, Q(3) accuracy of 0.83,
Matthews correlation coefficient of 0.74 and SOV for beta strand of 0.95. In
this self-consistency test the method predicted 83% of transmembrane residues
with correct exposure status. On an unseen, non-redundant test data set of 10
proteins, the 2-state and 3-state TMBHMM prediction accuracies are around 73%
and 72%, respectively, and are comparable to other methods from the literature.
The TMBHMM web server takes an amino acid sequence or a multiple sequence
alignment as an input and predicts the exposure status and the structural
topology as output. The TMBHMM web server is available under the tmbhmm tab at:
http://service.bioinformatik.uni-saarland.de/tmx-site/. Several computational methods exist for the identification of transmembrane beta
barrel proteins (TMBs) from sequence. Some of these methods also provide the
transmembrane (TM) boundaries of the putative TMBs. The aim of this study is to
(1) derive the propensities of the TM residues to be exposed to the lipid
bilayer and (2) to predict the exposure status (i.e. exposed to the bilayer or
hidden in protein structure) of TMB residues. Three novel propensity scales
namely, BTMC, BTMI and HTMI were derived for the TMB residues at the hydrophobic
core region of the outer membrane (OM), the lipid-water interface regions of the
OM, and for the helical membrane proteins (HMPs) residues at the lipid-water
interface regions of the inner membrane (IM), respectively. Separate propensity
scales were derived for monomeric and functionally oligomeric TMBs. The derived
propensities reflect differing physico-chemical properties of the respective
membrane bilayer regions and were employed in a computational method for the
prediction of the exposure status of TMB residues. Based on the these
propensities, the conservation indices and the frequency profile of the
residues, the transmembrane residues were classified into buried/exposed with an
accuracy of 77.91% and 80.42% for the residues at the membrane core and the
interface regions, respectively. The correlation of the derived scales with
different physico-chemical properties obtained from the AAIndex database are
also discussed. Knowledge about the residue propensities and burial status will
be useful in annotating putative TMBs with unknown structure. Predicting the solvent accessible surface area (ASA) of transmembrane (TM)
residues is of great importance for experimental researchers to elucidate
diverse physiological processes. TM residues fall into two major structural
classes (α-helix membrane protein and β-barrel membrane protein). The reported
solvent ASA prediction models were developed for these two types of TM residues
respectively. However, this prevents the general use of these methods because
one cannot determine which model is suitable for a given TM residue without
information of its type. To conquer this limitation, we developed a new
computational model that can be used for predicting the ASA of both TM α-helix
and β-barrel residues. The model was developed from 78 α-helix membrane protein
chains and 24 β-barrel membrane protein. Its prediction ability was evaluated by
cross validation method and its prediction result on an independent test set of
20 membrane protein chains. The results show that our model performs well for
both types of TM residues and outperforms other prediction model which was
developed for the specific type of TM residues. The prediction results also
proved that the random forest model incorporating conservation score is an
effective sequence-based computational approach for predicting the solvent ASA
of TM residues. MOTIVATION: Transmembrane β-barrels (TMBBs) are extremely important proteins
that play key roles in several cell functions. They cross the lipid bilayer with
β-barrel structures. TMBBs are presently found in the outer membranes of
Gram-negative bacteria and of mitochondria and chloroplasts. Loop exposure
outside the bacterial cell membranes makes TMBBs important targets for vaccine
or drug therapies. In genomes, they are not highly represented and are difficult
to identify with experimental approaches. Several computational methods have
been developed to discriminate TMBBs from other types of proteins. However, the
best performing approaches have a high fraction of false positive predictions.
RESULTS: In this article, we introduce a new machine learning approach for TMBB
detection based on N-to-1 Extreme Learning Machines that significantly
outperforms previous methods achieving a Matthews correlation coefficient of
0.82, a probability of correct prediction of 0.92 and a sensitivity of 0.73. Outer membrane β-barrel proteins differ from α-helical inner membrane proteins
in lipid environment, secondary structure, and the proposed processes of folding
and insertion. It is reasonable to expect that outer membrane proteins may
contain primary sequence information specific for their folding and insertion
behavior. In previous work, a depth-dependent insertion potential, E(z) , was
derived for α-helical inner membrane proteins. We have generated an equivalent
potential for TM β-barrel proteins. The similarities and differences between
these two potentials provide insight into unique aspects of the folding and
insertion of β-barrel membrane proteins. This potential can predict orientation
within the membrane and identify functional residues involved in intermolecular
interactions. We introduce a graph-theoretic model for predicting the supersecondary structure
of transmembrane β-barrel proteins--a particular class of proteins that performs
diverse important functions but it is difficult to determine their structure
with experimental methods. This ab initio model resolves the protein folding
problem based on pseudo-energy minimization with the aid of a simple
probabilistic filter. It also allows for determining structures whose barrel
follows a given permutation on the arrangement of β-strands, and allows for
rapidly discriminating the transmembrane β-barrels from other kinds of proteins.
The model is fairly accurate, robust and can be run very efficiently on PC-like
computers, thus proving useful for genome screening. BACKGROUND: Helical membrane proteins are vital for the interaction of cells
with their environment. Predicting the location of membrane helices in protein
amino acid sequences provides substantial understanding of their structure and
function and identifies membrane proteins in sequenced genomes. Currently there
is no comprehensive benchmark tool for evaluating prediction methods, and there
is no publication comparing all available prediction tools. Current benchmark
literature is outdated, as recently determined membrane protein structures are
not included. Current literature is also limited to global assessments, as
specialised benchmarks for predicting specific classes of membrane proteins were
not previously carried out.
DESCRIPTION: We present a benchmark server at
http://sydney.edu.au/pharmacy/sbio/software/TMH_benchmark.shtml that uses recent
high resolution protein structural data to provide a comprehensive assessment of
the accuracy of existing membrane helix prediction methods. The server further
allows a user to compare uploaded predictions generated by novel methods,
permitting the comparison of these novel methods against all existing methods
compared by the server. Benchmark metrics include sensitivity and specificity of
predictions for membrane helix location and orientation, and many others. The
server allows for customised evaluations such as assessing prediction method
performances for specific helical membrane protein subtypes.We report results
for custom benchmarks which illustrate how the server may be used for
specialised benchmarks. Which prediction method is the best performing method
depends on which measure is being benchmarked. The OCTOPUS membrane helix
prediction method is consistently one of the highest performing methods across
all measures in the benchmarks that we performed.
CONCLUSIONS: The benchmark server allows general and specialised assessment of
existing and novel membrane helix prediction methods. Users can employ this
benchmark server to determine the most suitable method for the type of
prediction the user needs to perform, be it general whole-genome annotation or
the prediction of specific types of helical membrane protein. Creators of novel
prediction methods can use this benchmark server to evaluate the performance of
their new methods. The benchmark server will be a valuable tool for researchers
seeking to extract more sophisticated information from the large and growing
protein sequence databases. |
Describe what is the usage of the Theatre software tool for genomic analysis. | Theatre is a web-based computing system designed for the comparative analysis of genomic sequences, especially with respect to motifs likely to be involved in the regulation of gene expression. Theatre is an interface to commonly used sequence analysis tools and biological sequence databases to determine or predict the positions of coding regions, repetitive sequences and transcription factor binding sites in families of DNA sequences. The information is displayed in a manner that can be easily understood and can reveal patterns that might not otherwise have been noticed. In addition to web-based output, Theatre can produce publication quality colour hardcopies showing predicted features in aligned genomic sequences. | |
How are CRM (cis-regulatory modules) defined? | Eukaryotic genes are often regulated by several transcription factors whose binding sites are tightly clustered and form cis-regulatory modules. | The binding of transcription factors to specific regulatory sequence elements is
a primary mechanism for controlling gene transcription. Eukaryotic genes are
often regulated by several transcription factors whose binding sites are tightly
clustered and form cis-regulatory modules. In this paper, we present a web
server, CREME, for identifying and visualizing cis-regulatory modules in the
promoter regions of a given set of potentially co-regulated genes. CREME relies
on a database of putative transcription factor binding sites that have been
annotated across the human genome using a library of position weight matrices
and evolutionary conservation with the mouse and rat genomes. A search algorithm
is applied to this data set to identify combinations of transcription factors
whose binding sites tend to co-occur in close proximity in the promoter regions
of the input gene set. The identified cis-regulatory modules are statistically
scored and significant combinations are reported and graphically visualized. Our
web server is available at http://creme.dcode.org. Mutations in a conserved non-coding region in intron 5 of the Lmbr1 locus, which
is 1 Mb away from the sonic hedgehog (Shh) coding sequence, are responsible for
mouse and human preaxial polydactyly with mirror-image digit duplications. In
the mouse mutants, ectopic Shh expression is observed in the anterior mesenchyme
of limb buds. Furthermore, a transgenic reporter gene flanked with this
conserved non-coding region shows normal polarized expression in mouse limb
buds. This conserved sequence has therefore been proposed to act as a
long-range, cis-acting regulator of limb-specific Shh expression. Previous
phylogenetic studies have also shown that this sequence is highly conserved
among tetrapods, and even in teleost fishes. Paired fins of teleost fishes and
tetrapod limbs have evolved from common ancestral appendages, and polarized Shh
expression is commonly observed in fins. In this study, we first show that this
conserved sequence motif is also physically linked to the Shh coding sequence in
a teleost fish, the medaka, by homology search of a newly available genomic
sequence database. Next, we show that deletion of this conserved intronic
sequence by targeted mutation in the mouse results in a complete loss of Shh
expression in the limb bud and degeneration of skeletal elements distal to the
stylopod/zygopod junction. This sequence contains a major limb-specific Shh
enhancer that is necessary for distal limb development. These results suggest
that the conserved intronic sequence evolved in a common ancestor of fishes and
tetrapods to control fin and limb development. Lack of knowledge about how regulatory regions evolve in relation to their
structure-function may limit the utility of comparative sequence analysis in
deciphering cis-regulatory sequences. To address this we applied reverse
genetics to carry out a functional genetic complementation analysis of a
eukaryotic cis-regulatory module-the even-skipped stripe 2 enhancer-from four
Drosophila species. The evolution of this enhancer is non-clock-like, with
important functional differences between closely related species and functional
convergence between distantly related species. Functional divergence is
attributable to differences in activation levels rather than spatiotemporal
control of gene expression. Our findings have implications for understanding
enhancer structure-function, mechanisms of speciation and computational
identification of regulatory modules. Transcription regulation is controlled by coordinated binding of one or more
transcription factors in the promoter regions of genes. In many species,
especially higher eukaryotes, transcription factor binding sites tend to occur
as homotypic or heterotypic clusters, also known as cis-regulatory modules. The
number of sites and distances between the sites, however, vary greatly in a
module. We propose a statistical model to describe the underlying cluster
structure as well as individual motif conservation and develop a Monte Carlo
motif screening strategy for predicting novel regulatory modules in upstream
sequences of coregulated genes. We demonstrate the power of the method with
examples ranging from bacterial to insect and human genomes. To facilitate the analysis of gene regulatory regions of the mouse genome, we
developed a CisView (http://lgsun.grc.nia.nih.gov/cisview), a browser and
database of genome-wide potential transcription factor binding sites (TFBSs)
that were identified using 134 position-weight matrices and 219 sequence
patterns from various sources and were presented with the information about
sequence conservation, neighboring genes and their structures, GO annotations,
protein domains, DNA repeats and CpG islands. Analysis of the distribution of
TFBSs revealed that many TFBSs (N = 145) were over-represented near
transcription start sites. We also identified potential cis-regulatory modules
(CRMs) defined as clusters of conserved TFBSs in the entire mouse genome. Out of
739 074 CRMs, 157 442 had a significantly higher regulatory potential score than
semi-random sequences generated with a 3rd-order Markov process. The CisView
browser provides a user-friendly computer environment for studying transcription
regulation on a whole-genome scale and can also be used for interpreting
microarray experiments and identifying putative targets of transcription
factors. The larval nervous system of the ascidian Ciona intestinalis exhibits an
abstract form of the vertebrate nervous system. The Ci-Galphai1 gene, which
encodes a G-protein alpha subunit, is specifically expressed in distinct sets of
neurons in C. intestinalis larvae, including papillar neurons of the adhesive
organ, ocellus photoreceptor cells, and cholinergic and GABAergic neurons in the
central nervous system (CNS). A GFP reporter gene driven by the 4.2-kb 5'
flanking region of Ci-Galphai1 recapitulated the endogenous gene expression
patterns. Comparative genomic analysis of the Galphai1 gene between C.
intestinalis and Ciona savignyi identified an 87-bp highly conserved non-coding
sequence located between -3176 and -3090 bp upstream of the gene. Deletion of
this conserved upstream sequence resulted in the complete loss of reporter
expression in the central nervous system, while reporter expression in the
adhesive organ and mesenchyme cells remained unaffected. The conserved upstream
sequence can activate gene expression from basal promoters in the brain vesicle,
although it requires additional cis-regulatory sequences to fully activate the
CNS-specific gene expression. These results suggest that different types of
central neurons share a common transcriptional activation mechanism that is
different from that of papillar neurons. The notochord is a defining feature of the chordate body plan. Experiments in
ascidian, frog and mouse embryos have shown that co-expression of Brachyury and
FoxA class transcription factors is required for notochord development. However,
studies on the cis-regulatory sequences mediating the synergistic effects of
these transcription factors are complicated by the limited knowledge of
notochord genes and cis-regulatory modules (CRMs) that are directly targeted by
both. We have identified an easily testable model for such investigations in a
155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM
contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA
(Ci-FoxA-a). By combining point mutation analysis and misexpression experiments,
we demonstrate that binding of both transcription factors to this CRM is
necessary and sufficient to activate transcription. To gain insights into the
cis-regulatory criteria controlling its activity, we investigated the
organization of the transcription factor binding sites within the 155-bp CRM.
The 155-bp sequence contains two Ci-Bra binding sites with identical core
sequences but opposite orientations, only one of which is required for enhancer
activity. Changes in both orientation and spacing of these sites substantially
affect the activity of the CRM, as clusters of identical sites found in the
Ciona genome with different arrangements are unable to activate transcription in
notochord cells. This work presents the first evidence of a synergistic
interaction between Brachyury and FoxA in the activation of an individual
notochord CRM, and highlights the importance of transcription factor binding
site arrangement for its function. Computational methods attempting to identify instances of cis-regulatory modules
(CRMs) in the genome face a challenging problem of searching for potentially
interacting transcription factor binding sites while knowledge of the specific
interactions involved remains limited. Without a comprehensive comparison of
their performance, the reliability and accuracy of these tools remains unclear.
Faced with a large number of different tools that address this problem, we
summarized and categorized them based on search strategy and input data
requirements. Twelve representative methods were chosen and applied to predict
CRMs from the Drosophila CRM database REDfly, and across the human ENCODE
regions. Our results show that the optimal choice of method varies depending on
species and composition of the sequences in question. When discriminating CRMs
from non-coding regions, those methods considering evolutionary conservation
have a stronger predictive power than methods designed to be run on a single
genome. Different CRM representations and search strategies rely on different
CRM properties, and different methods can complement one another. For example,
some favour homotypical clusters of binding sites, while others perform best on
short CRMs. Furthermore, most methods appear to be sensitive to the composition
and structure of the genome to which they are applied. We analyze the principal
features that distinguish the methods that performed well, identify weaknesses
leading to poor performance, and provide a guide for users. We also propose key
considerations for the development and evaluation of future CRM-prediction
methods. How multiple developmental cues are integrated on cis-regulatory modules (CRMs)
for cell fate decisions remains uncertain. The Spemann-Mangold organizer in
Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1,
Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation
and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated
by the aforementioned transcription factors through CRMs conserved between X.
laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors
revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the
late blastula stage, and subsequently bound by all five factors at the gastrula
stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among
others, persists, which corresponds to their co-expression in the prechordal
plate. Based on these data, together with detailed expression pattern analysis,
we propose a new model of stepwise formation of the organizer, in which (1)
maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then
(2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the
dorsal endodermal and mesodermal regions where all these genes are co-expressed;
and (3) these two regions are combined at the gastrula stage to form the
organizer. Thus, the in vivo dynamics of multiple transcription factors
highlight their roles in the initiation and maintece of gene expression, and
also reveal the stepwise integration of maternal, Nodal and Wnt signaling on
CRMs of organizer genes to generate the organizer. Somites form by an iterative process from unsegmented, presomitic mesoderm
(PSM). Notch pathway components, such as deltaC (dlc) have been shown to play a
role in this process, while the T-box transcription factors Ntla and Tbx16
regulate somite formation upstream of this by controlling supply and movement of
cells into the PSM during gastrulation and tailbud outgrowth. In this work, we
report that Ntla and Tbx16 play a more explicit role in segmentation by directly
regulating dlc expression. In addition we describe a cis-regulatory module (CRM)
upstream of dlc that drives expression of a reporter in the tailbud, PSM and
somites during somitogenesis. This CRM is bound by both Ntla and Tbx16 at a
cluster of T-box binding sites, which are required in combination for activation
of the CRM. BACKGROUND: The identification of transcription factor binding sites (TFBSs) and
cis-regulatory modules (CRMs) is a crucial step in studying gene expression, but
the computational method attempting to distinguish CRMs from NCNRs still remains
a challenging problem due to the limited knowledge of specific interactions
involved.
METHODS: The statistical properties of cis-regulatory modules (CRMs) are
explored by estimating the similar-word set distribution with overrepresentation
(Z-score). It is observed that CRMs tend to have a thin-tail Z-score
distribution. A new statistical thin-tail test with two thinness coefficients is
proposed to distinguish CRMs from non-coding non-regulatory regions (NCNRs).
RESULTS: As compared with the existing fluffy-tail test, the first thinness
coefficient is designed to reduce computational time, making the novel thin-tail
test very suitable for long sequences and large database analysis in the
post-genome time and the second one to improve the separation accuracy between
CRMs and NCNRs. These two thinness coefficients may serve as valuable filtering
indexes to predict CRMs experimentally.
CONCLUSIONS: The novel thin-tail test provides an efficient and effective means
for distinguishing CRMs from NCNRs based on the specific statistical properties
of CRMs and can guide future experiments aimed at finding new CRMs in the
post-genome time. BACKGROUND: In eukaryotes, transcriptional regulation is usually mediated by
interactions of multiple transcription factors (TFs) with their respective
specific cis-regulatory elements (CREs) in the so-called cis-regulatory modules
(CRMs) in DNA. Although the knowledge of CREs and CRMs in a genome is crucial to
elucidate gene regulatory networks and understand many important biological
phenomena, little is known about the CREs and CRMs in most eukaryotic genomes
due to the difficulty to characterize them by either computational or
traditional experimental methods. However, the exponentially increasing number
of TF binding location data produced by the recent wide adaptation of chromatin
immunoprecipitation coupled with microarray hybridization (ChIP-chip) or
high-throughput sequencing (ChIP-seq) technologies has provided an unprecedented
opportunity to identify CRMs and CREs in genomes. Nonetheless, how to
effectively mine these large volumes of ChIP data to identify CREs and CRMs at
nucleotide resolution is a highly challenging task.
RESULTS: We have developed a novel graph-theoretic based algorithm DePCRM for
genome-wide de novo predictions of CREs and CRMs using a large number of ChIP
datasets. DePCRM predicts CREs and CRMs by identifying overrepresented
combinatorial CRE motif patterns in multiple ChIP datasets in an effective way.
When applied to 168 ChIP datasets of 56 TFs from D. melanogaster, DePCRM
identified 184 and 746 overrepresented CRE motifs and their combinatorial
patterns, respectively, and predicted a total of 115,932 CRMs in the genome. The
predictions recover 77.9% of known CRMs in the datasets and 89.3% of known CRMs
containing at least one predicted CRE. We found that the putative CRMs as well
as CREs as a whole in a CRM are more conserved than randomly selected sequences.
CONCLUSION: Our results suggest that the CRMs predicted by DePCRM are highly
likely to be functional. Our algorithm is the first of its kind for de novo
genome-wide prediction of CREs and CRMs using larger number of transcription
factor ChIP datasets. The algorithm and predictions will hopefully facilitate
the elucidation of gene regulatory networks in eukaryotes. All the predicted
CREs, CRMs, and their target genes are available at
http://bioinfo.uncc.edu/mniu/pcrms/www/. |
Which is the main regulatory molecule of SERCA2A function in the cardiac muscle? | SERCA2a activity is regulated by phosphorylation of another SR protein, Phospholamban (PLN). Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca(2+). | Dilated cardiomyopathy and end-stage heart failure result in multiple defects in
cardiac excitation-contraction coupling. Via complementation of a genetically
based mouse model of dilated cardiomyopathy, we now provide evidence that
progressive chamber dilation and heart failure are dependent on a Ca2+ cycling
defect in the cardiac sarcoplasmic reticulum. The ablation of a muscle-specific
sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) inhibitor, phospholamban, rescued
the spectrum of phenotypes that resemble human heart failure. Inhibition of
phospholamban-SERCA2a interaction via in vivo expression of a phospholamban
point mutant domitly activated the contractility of ventricular muscle cells.
Thus, interfering with phospholamban-SERCA2a interaction may provide a novel
therapeutic approach for preventing the progression of dilated cardiomyopathy. Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated
region of a protein kinase gene (DMPK). Cardiovascular disease is one of the
most prevalent causes of death in DM patients. Electrophysiological studies in
cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is
critical to the modulation of cardiac contractility and to the maintece of
proper cardiac conduction activity. However, there are no data regarding the
molecular signaling pathways involved in DM heart failure. Here we show that
DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic
reticulum (SR) where it colocalizes with the ryanodine receptor and
phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor.
Coimmunoprecipitation studies showed that DMPK and PLN can physically associate.
Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A
DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice
demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice
compared with wild-type mice both in vitro and in vivo. Finally, we show that
Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice.
Together, our data suggest the existence of a novel regulatory DMPK pathway for
cardiac contractility and provide a molecular mechanism for DM heart pathology. Intracellular calcium is a major coordinator of numerous aspects of cellular
physiology, including muscle contractility and cell survival. In cardiac muscle,
aberrant Ca(2+) cycling has been implicated in a range of pathological
conditions including cardiomyopathies and heart failure. The sarco(endo)plasmic
reticulum Ca(2+) transport adenosine triphosphatase (SERCA2a) and its regulator
phospholamban (PLN) have a central role in modulating Ca(2+) homeostasis and,
therefore, cardiac function. Herein, we discuss the mechanisms through which
SERCA2a and PLN control cardiomyocyte function in health and disease. Emphasis
is placed on our newly identified PLN-binding partner HS-1-associated protein
X-1 (HAX-1), which has an anti-apoptotic function and presents with numerous
similarities to Bcl-2. Recent evidence indicates that proteins of the Bcl-2
family can influence ER Ca(2+) content, a critical determit of cellular
sensitivity to apoptosis. The discovery of the PLN/HAX-1 interaction therefore
unveils an important new link between Ca(2+) homeostasis and cell survival, with
significant therapeutic potential. Sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) transports Ca2+ into the SR,
decreasing the cytosolic Ca2+ during relaxation and increasing the SR Ca2+
available for contraction. SERCA2a activity is regulated by phosphorylation of
another SR protein: Phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a.
Phosphorylation of PLN by either cAMP or cGMP-dependent protein kinase at Ser16
or the Ca2+-calmodulin-dependent protein kinase (CaMKII), at Thr17, relieves
this inhibition, increasing SR Ca2+ uptake and SR Ca2+ load. Thus, PLN is a
major player in the regulation of myocardial relaxation and contractility. This
review will examine the main aspects of the role of CaMKII and Thr17 site of
PLN, on different pathophysiological conditions: acidosis, ischemia/reperfusion
(I/R) and heart failure (HF). Whereas CaMKII-activation and PLN phosphorylation
contribute to the functional recovery during acidosis and stunning, CaMKII
results detrimental in the irreversible I/R injury, producing apoptosis and
necrosis. Phosphorylation of Thr17 residue of PLN and CaMKII activity vary in
the different models of HF. The possible role of these changes in the depressed
cardiac function of HF will be discussed. Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in
cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the
enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding
and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2
conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation
that binds two Ca(2+) (at sites I and II) with high affinity and is required for
ATP hydrolysis. Here we determined whether Ca(2+) binding to site I, site II, or
both sites is sufficient to dissociate PLB from the Ca(2+) pump. Seven SERCA2a
mutants with amino acid substitutions at Ca(2+)-binding site I (E770Q, T798A,
and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q)
were made, and the effects of Ca(2+) on N30C-PLB cross-linking to Lys(328) of
SERCA2a were measured. In agreement with earlier reports with the skeletal
muscle Ca(2+)-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP
in the presence of Ca(2+); however, all were phosphorylatable by P(i) to form
E2P. Ca(2+) inhibition of E2P formation was observed only in SERCA2a mutants
retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB
and each Ca(2+)-ATPase mutant was observed in the absence of Ca(2+).
Importantly, however, micromolar Ca(2+) inhibited PLB cross-linking only to
mutants retaining a functional Ca(2+)-binding site I. The dynamic equilibrium
between Ca(2+) pumps and N30C-PLB was retained by all mutants, demonstrating
normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody.
From these results we conclude that site I is the key Ca(2+)-binding site
regulating the physical association between PLB and SERCA2a. Phospholamban (PLN), the reversible inhibitor of the sarco(endo)plasmic
reticulum Ca(2+)-ATPase (SERCA2a), is a key regulator of myocyte Ca(2+) cycling
with a significant role in heart failure. We previously showed that the single
amino acid difference between human and mouse PLN results in increased
inhibition of Ca(2+) cycling and cardiac remodeling and attenuated stress
responses in transgenic mice expressing the human PLN (hPLN) in the null
background. Here we dissect the molecular and electrophysiological processes
triggered by the superinhibitory hPLN in the mouse. Using a multidisciplinary
approach, we performed global gene expression analysis, electrophysiology, and
mathematical simulations on hPLN mice. We identified significant changes in a
series of Na(+) and K(+) homeostasis genes/proteins (including Kcnd2, Scn9a,
Slc8a1) and ionic conductance (including L-type Ca(2+) current, Na(+)/Ca(2+)
exchanger, transient outward K(+) current). Simulation analysis suggests that
this electrical remodeling has a critical role in rescuing cardiac function by
improving sarcoplasmic reticulum Ca(2+) load and overall Ca(2+) dynamics.
Furthermore, multiple structural and transcription factor gene expression
changes indicate an ongoing structural remodeling process, favoring hypertrophy
and myogenesis while suppressing apoptosis and progression to heart failure. Our
findings expand current understanding of the hPLN function and provide
additional insights into the downstream implications of SERCA2a superinhibition
in the mammalian heart. Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR
Ca-transport and Ca-release, is a key and universal characteristic of human and
experimental heart failure. These SR processes are regulated by multimeric
protein complexes, including protein kinases and phosphatases as well as their
anchoring and regulatory subunits that fine-tune Ca-handling in specific SR
sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and
its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an
inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or
calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory
effects. Recent studies identified additional regulatory proteins, associated
with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of
protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the
HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal
histidine-rich calcium binding protein (HRC) has been shown to interact with
both SERCA2a and triadin. Notably, there is physical and direct interaction
between these protein players, mediating a fine-cross talk between SR Ca-uptake,
storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA
interactome does not only impact cardiomyocyte contractility, but also survival
and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes
modulate their activity and interactions with other protein partners, resulting
in depressed contractility and accelerated remodeling. These genetic variants
may serve as potential prognostic or diagnostic markers in cardiac
pathophysiology. |
Which gene is responsible for the development of the Mowat-Wilson syndrome? | Mowat-Wilson syndrome is a genetic disease caused by heterozygous mutations or deletions of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. | We report a girl who had Hirschsprung disease in association with distinct
facial appearance, microcephaly, agenesis of the corpus callosum and mental
retardation (Mowat-Wilson syndrome). Mutation analysis of the zinc finger homeo
box 1 B (ZFHX1 B) gene revealed a de novo 7 bp deletion (TGGCCCC) at nucleotide
1773 (1773 delTGGCCCC) resulting in a frameshift and leading to a termination
codon at amino acid residue 604 (604 X) in exon 8 C. The zinc finger homeo box 1
B (Smad interacting protein-1) is a transcription corepressor of Smad target
genes with functions in the patterning of neural crest derived cells, CNS, and
midline structures. Mutations in ZFHX1 B can lead to neurological disorders in
addition to dysmorphic features, megacolon, and other malformations. ZFHX1B encodes Smad-interacting protein 1, a transcriptional corepressor
involved in the transforming growth factors beta (TGFbeta) signaling pathway.
ZFHX1B mutations cause a complex developmental phenotype characterized by severe
mental retardation (MR) and multiple congenital defects. We compared the
distribution of ZFHX1B transcripts during mouse and human embryogenesis as well
as in adult mice and humans. This showed that this gene is strongly transcribed
at an early stage in the developing peripheral and central nervous systems of
both mice and humans, in all neuronal regions of the brains of 25-week human
fetuses and adult mice, and at varying levels in numerous nonneural tissues.
Northern blot analysis suggested that ZFHX1B undergoes tissue-specific
alternative splicing in both species. These results strongly suggest that ZFHX1B
determines the transcriptional levels of target genes in various tissues through
the combinatorial interactions of its isoforms with different Smad proteins.
Thus, as well as causing neural defects, ZFHX1B mutations may also cause other
malformations. Hypospadias, when the urethra opens on the ventral side of the penis, is a
common malformation seen in about 3 per 1,000 male births. It is a complex
disorder associated with genetic and environmental factors and can be part of
genetic syndromes. Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly
syndrome characterized by a distinct facial phenotype, Hirschsprung disease,
microcephaly and mental retardation. It is caused by mutations in the zinc
finger homeo box 1B gene, ZFHX1B (SIP1). To date, 68 deletion/mutation-positive
cases have been reported. Genitourinary anomalies are common in MWS. Here we
report that hypospadias is common in males with this syndrome. In 39 patients
where this information was available, hypospadias was present in 46% of patients
(18/39). In the 3 Italian male cases reported here, hypospadias was always
present. MWS should be considered by endocrinologists in patients with
hypospadias associated with developmental delays/mental retardation, in
particular in the presence of a distinct facial phenotype. Mowat-Wilson syndrome (MWS) is a rare mental retardation-multiple congenital
anomalies syndrome associated with typical facial dysmorphism. Patients can show
a variety of other anomalies like short stature, microcephaly, Hirschsprung
disease, malformations of the brain, seizures, congenital heart defects and
urogenital anomalies. Mutations leading to haploinsufficiency of the ZFHX1B gene
have been described as the underlying cause of this condition. We report on the
clinical findings in a 2(1/2)-year-old boy with some aspects out of the
MWS-spectrum in addition to unusual anomalies and a novel missense mutation in
the ZFHX1B gene. Mowat-Wilson syndrome (MWS) is a relatively newly described multiple congenital
anomaly/mental retardation syndrome. Haploinsufficiency of a gene termed ZFHX1B
(also known as SIP1) on chromosome 2 is responsible for this condition, and
clinical genetic testing for MWS recently became available. The majority of
reports in the literature originate from Northern Europe and Australia. Here we
report our clinical experience with 12 patients diagnosed with MWS within a
2-year period of time in the United States, with particular emphasis on clinical
characteristics and management strategies. Individuals with this condition have
characteristic facial features, including microcephaly, hypertelorism, medially
flared and broad eyebrows, prominent columella, pointed chin, and uplifted
earlobes, which typically prompt the clinician to consider the diagnosis.
Medical issues in our cohort of patients included seizures (75%) with no
predeliction for any particular seizure type; agenesis of the corpus callosum
(60% of our patients studied); congenital heart defects (75%), particularly
involving the pulmonary arteries and/or valves; hypospadias (55% of males);
severely impaired or absent speech (100% of individuals over 1 year of age) with
relatively spared receptive language; and Hirschsprung disease (50%) or chronic
constipation (25%). The incidence of MWS is unknown, but based on the number of
patients identified in a short period of time within the US, it is likely
greatly under recognized. MWS should be considered in any individual with
severely impaired or absent speech, especially in the presence of seizures and
anomalies involving the pulmonary arteries (particularly pulmonary artery sling)
or pulmonary valves. Mowat-Wilson syndrome (MWS) is a recently delineated mental retardation
(MR)-multiple congenital anomaly syndrome, characterized by typical facies,
severe MR, epilepsy, and variable congenital malformations, including
Hirschsprung disease (HSCR), genital anomalies, congenital heart disease (CHD),
and agenesis of the corpus callosum (ACC). It is caused by de novo heterozygous
mutations or deletions of the ZFHX1B gene located at 2q22. ZFHX1B encodes
Smad-interacting protein-1 (SMADIP1 or SIP1), a transcriptional corepressor
involved in the transforming growth factor-beta signaling pathway. It is a
highly evolutionarily conserved gene, widely expressed in embryological
development. Over 100 mutations have been described in patients with clinically
typical MWS, who almost always have whole gene deletions or truncating mutations
(nonsense or frameshift) of ZFHX1B, suggesting that haploinsufficiency is the
basis of MWS pathology. No obvious genotype-phenotype correlation could be
identified so far, but atypical phenotypes have been reported with missense or
splice mutations in the ZFHX1B gene. In this work we describe 40 novel mutations
and we summarize the various mutational reports published since the
identification of the causative gene. Mowat-Wilson syndrome is a recently delineated autosomal domit developmental
anomaly, whereby heterozygous mutations in the ZFHX1B gene cause mental
retardation, delayed motor development, epilepsy and a wide spectrum of
clinically heterogeneous features, suggestive of neurocristopathies at the
cephalic, cardiac and vagal levels. However, our understanding of the etiology
of this condition at the cellular level remains vague. This study presents the
Zfhx1b protein expression domain in mouse embryos and correlates this with a
novel mouse model involving a conditional mutation in the Zfhx1b gene in neural
crest precursor cells. These mutant mice display craniofacial and
gastrointestinal malformations that show resemblance to those found in human
patients with Mowat-Wilson syndrome. In addition to these clinically recognized
alterations, we document developmental defects in the heart, melanoblasts and
sympathetic and parasympathetic anlagen. The latter observations in our mouse
model for Mowat-Wilson suggest a hitherto unknown role for Zfhx1b in the
development of these particular neural crest derivatives, which is a set of
observations that should be acknowledged in the clinical management of this
genetic disorder. Mowat-Wilson syndrome is a genetic condition characterized by a recognizable
facial phenotype in addition to moderate to severe cognitive disability with
severe speech impairment and variable multiple congenital anomalies. The
anomalies may include Hirschsprung disease, heart defects, structural eye
anomalies including microphthalmia, agenesis of the corpus callosum, and
urogenital anomalies. Microcephaly, seizure disorder and constipation are
common. All typical cases result from haploinsufficiency of the ZEB2 (also known
as ZFHX1B or SIP-1) gene, with over 100 distinct mutations now described.
Approximately 80% of patients have a nonsense or frameshift mutation detectable
by sequencing, with the rest having gross deletions necessitating a dosage
sensitive assay. Here we report on the results of comprehensive molecular
testing for 27 patients testing positive for MWS. Twenty-one patients had a
nonsense, frameshift, or splice site mutation identified by sequencing; 14 of
which localized to exon 8 and 17 of which are novel. Six patients had deletions
in the ZEB2 gene, including two novel partial gene deletions. This report, the
first such analysis in North American patients, adds to the growing list of both
novel pathogenic mutations associated with MWS, as well as other variants in the
ZEB2 gene. In addition, we suggest an economical testing strategy. Rubinstein-Taybi syndrome (RSTS) is a rare autosomal domit disorder
characterised by facial dysmorphisms, growth and psychomotor development delay,
and skeletal defects. The known genetic causes are point mutations or deletions
of the CREBBP (50-60%) and EP300 (5%) genes. To detect chromosomal
rearrangements indicating novel positional candidate RSTS genes, we used a-CGH
to study 26 patients fulfilling the diagnostic criteria for RSTS who were
negative at fluorescence in situ hybridisation analyses of the CREBBP and EP300
regions, and direct sequencing analyses of the CREBBP gene. We found seven
imbalances (27%): four de novo and three inherited rearrangements not reported
among the copy number variants. A de novo 7p21.1 deletion of 500 kb included the
TWIST1 gene, a suggested candidate for RSTS that is responsible for the
Saethre-Chotzen syndrome, an entity that enters in differential diagnosis with
RSTS. A similar issue of differential diagnosis was raised by a large 4.3 Mb
2q22.3q23.1 deletion encompassing ZEB2, the gene responsible for the
Mowat-Wilson syndrome, whose signs may overlap with RSTS. Positional candidate
genes could not be sought in the remaining pathogenetic imbalances, because of
the size of the involved region (a 9 Mb 2q24.3q31.1 deletion) and/or the
relative paucity of suitable genes (a 5 Mb 3p13p12.3 duplication). One of the
inherited rearrangements, the 17q11.2 379Kb duplication, represents the
reciprocal event of the deletion underlying an overgrowth syndrome, both being
mediated by the NF1-REP-P1 and REP-P2 sub-duplicons. The contribution of this
and the other detected CNVs to the clinical RSTS phenotype is difficult to
assess. Mowat-Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive
facial gestalt-multiple congenital anomaly syndrome, commonly associating
microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects,
urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de
novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to
haplo-insufficiency through premature stop codons or large gene deletions. Only
three missense mutations have been reported so far; none of which resides in a
known functional domain of ZEB2. In this study, we report and analyze the
functional consequences of three novel missense mutations, p.Tyr1055Cys,
p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger
(C-ZF) domain of ZEB2. Patients' phenotype included the facial gestalt of MWS
and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies
showed that all the three mutations prevented binding and repression of the
E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the
zebrafish morphant technology, we performed rescue experiments using wild-type
(WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue,
correlating with the severity of patients' phenotype, was observed. Our data
provide evidence that these missense mutations cause a partial loss of function
of ZEB2, suggesting that its role is not restricted to repression of E-cadherin.
Functional domains other than C-ZF may play a role in early embryonic
development. Finally, these findings broaden the clinical spectrum of ZEB2
mutations, indicating that MWS ought to be considered in patients with lesser
degrees of ID and a suggestive facial gestalt, even in the absence of congenital
malformation. Mowat-Wilson syndrome (MWS) is a genetic disease caused by heterozygous
mutations or deletions of the ZEB2 gene rarely diagnosed prenatally and with
little fetal description reported. It is mainly characterized by
moderate-to-severe intellectual disability, epilepsy, facial dysmorphism and
various malformations including Hirschsprung disease and corpus callosum
anomalies. Here we report a fetal case of MWS well described, suspected at
standard autopsy. The association of a corpus callosum hypoplasia with a
histological Hirschsprung disease and a typical facial gestalt allowed the
guiding of genetic testing. Classical fetopathological examination still keeps
indications in cases of syndromic association in the era of virtual autopsy. Individuals with Mowat-Wilson syndrome (MWS; OMIM#235730) have characteristic
facial features, a variety of congenital anomalies such as Hirschsprung disease,
and intellectual disabilities caused by mutation or deletion of ZEB2 gene. This
deletion or cytogenetic abnormality has been reported primarily from Europe,
Australia and the United States, but not in Korea. Here we report a patient with
characteristic facial features of MWS, developmental delay and spasticity. High
resolution microarray analysis revealed 0.9 Mb deletion of 2q22.3 involving two
genes: ZEB2 and GTDC1. This case shows the important role of high resolution
microarray in patients with unexplained psychomotor retardation and/or facial
dysmorphism. Knowledge about the most striking clinical signs and implementation
of effective molecular tests like microarray could significantly increase the
detection rate of new cases of MWS in Korea. This is the first reported case of
MWS in Korea. Mowat-Wilson syndrome is a genetic disease caused by heterozygous mutations or
deletions of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. The syndrome
is characterized by typical facial features, moderate-to-severe mental
retardation, epilepsy and variable congenital malformations, including
Hirschsprung disease, genital anomalies, congenital heart disease, agenesis of
the corpus callosum, and eye defects. The prevalence of Mowat-Wilson syndrome is
currently unknown, but it seems that Mowat-Wilson syndrome is underdiagnosed,
particularly in patients without Hirschsprung disease. We report here the first
Egyptian case of Mowat-Wilson syndrome who was conceived by intracytoplasmic
sperm injection. The patient manifested bilateral sensorineural hearing loss--a
new feature not previously reported in cases of Mowat-Wilson syndrome. This
report describes the first Egyptian patient of Mowat-Wilson syndrome who was
conceived after intracytoplasmic sperm injection, and provides a new evidence
for the inclusion of deafness among the congenital defects of the syndrome. |
How is active neurotoxin of Clostridium botulinum detected? | Active neurotoxin of Clostridium botulinum can be detected by:
mouse lethality assay
by mass spectrometry
bioassay
differentiated cell models
peptide cleavage assay
FDC (functional dual coating) microtitre plate immuno-biochemical assay
endopeptidase activity monitored via UV-Visible spectroscopy | Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular
proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G)
exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid,
mass spectrometry-based method (Endopep-MS) to detect and differentiate active
BoNTs A, B, E, and F. This method uses the highly specific protease activity of
the toxins with target peptides specific for each toxin serotype. The product
peptides derived from the endopeptidase activities of BoNTs are detected by
matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In
buffer, this method can detect toxin equivalents of as little as 0.01 mouse
lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A
high-performance liquid chromatography-tandem mass spectrometry method for
quantifying active toxin, where the amount of toxin can be correlated to the
amount of product peptides, is also described. The World Health Organization (WHO) and U.S. Centers for Disease Control and
Prevention (CDC) have labeled botulinum toxins as a high priority biological
agent that may be used in terrorist attacks against food supplies. Due to this
threat there is an increased need to develop fast and effective methods to
detect active botulinum neurotoxins (BoNTs). This study reports the successful
use of an enzymatic assay employing an internally quenched fluorogenic peptide
as a fast, simple and inexpensive alternative to the mouse bioassay. In less
than 15 min the assay can detect 0.25 nM BoNT-A in liquid food samples. The
detection level is far below the adult human lethal oral dose of 70 microg of
toxin. Immunomagnetic beads coated with IgG monoclonal antibodies that target
the toxin heavy chain can concentrate the toxin without neutralizing its
enzymatic activity, overcoming matrix effects caused by endogenous protease
inhibitors and peptidases. This fast and effective assay system could be used
for large scale screening to detect BoNT-A. Human induced pluripotent stem cells (hiPSC) hold great promise for providing
various differentiated cell models for in vitro toxigenicity testing. For
Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies,
several cell models currently exist, but none examine toxin function with
species-specific relevance while exhibiting high sensitivity. The most sensitive
cell models to date are mouse or rat primary cells and neurons derived from
mouse embryonic stem cells, both of which require significant technical
expertise for culture preparation. This study describes for the first time the
use of hiPSC-derived neurons for BoNT detection. The neurons used in this study
were differentiated and cryopreserved by Cellular Dynamics International
(Madison, WI) and consist of an almost pure pan-neuronal population of
predomitly gamma aminoisobutyric acidergic and glutamatergic neurons. Western
blot and quantitative PCR data show that these neurons express all the necessary
receptors and substrates for BoNT intoxication. BoNT/A intoxication studies
demonstrate that the hiPSC-derived neurons reproducibly and quantitatively
detect biologically active BoNT/A with high sensitivity (EC(50) ∼0.3 U).
Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A
complex was demonstrated, and BoNT/A specificity was confirmed through antibody
protection studies. A direct comparison of BoNT detection using primary rat
spinal cord cells and hiPSC-derived neurons showed equal or increased
sensitivity, a steeper dose-response curve and a more complete SNARE protein
target cleavage for hiPSC-derived neurons. In summary, these data suggest that
neurons derived from hiPSCs provide an ideal and highly sensitive platform for
BoNT potency determination, neutralizing antibody detection and for mechanistic
studies. Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the
causative agent of botulism, and are widely used as valuable pharmaceuticals.
The BoNTs are modular proteins consisting of a heavy chain and a light chain
linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a
multi-step process including specific cell binding, endocytosis, conformational
change in the endosome, translocation of the enzymatic light chain into the
cells cytosol, and SNARE target cleavage. The quantitative and reliable potency
determination of fully functional BoNTs produced as active pharmaceutical
ingredient (API) requires an assay that considers all steps in the intoxication
pathway. The in vivo mouse bioassay has for years been the 'gold standard' assay
used for this purpose, but it requires the use of large numbers of mice and thus
causes associated costs and ethical concerns. Cell-based assays are currently
the only in vitro alternative that detect fully functional BoNTs in a single
assay and have been utilized for years for research purposes. Within the last
5 years, several cell-based BoNT detection assays have been developed that are
able to quantitatively determine BoNT potency with similar or greater
sensitivity than the mouse bioassay. These assays now offer an alternative
method for BoNT potency determination. Such quantitative and reliable BoNT
potency determination is a crucial step in basic research, in the development of
pharmaceutical BoNTs, and in the quantitative detection of neutralizing
antibodies. Clostridium botulinum type A toxin is the most prevalent cause of naturally
occurring outbreaks of human botulism in the world. The active dichain
neurotoxin molecule is composed of a heavy chain (H-chain) of ~100 kDa with the
carboxy-terminal end consisting of a receptor-binding (HC) domain, while the
amino-terminal (HN) domain is linked by a critical disulfide bond to a light
chain (L-chain) of ~50 kDa. Although the mouse bioassay (MBA) is traditionally
used to confirm the presence of toxin in serum or food, its sensitivity is
insufficient to detect low toxin levels in approximately 30 to 60 % of botulism
patients. A novel FDC (functional dual coating) microtitre plate
immuno-biochemical assay, which quantifies botulinum toxicity by measuring the
HC domain linked with L-chain endopeptidase activity, was modified to allow
human serum (lysed or unlysed) to be tested without interference from the
matrix, with toxin detection down to 0.03 mouse LD50 per ml serum or 0.13 pg
ml(-1) using just 100 µl of clinical samples. The assay was specific for type A
toxin and could additionally be applied to whole blood and food samples. Low
levels of 1 to 2 mouse LD50 per ml serum of type A toxin were quantified for the
first time using the modified FDC assay in two severely intoxicated UK patients
who required mechanical ventilation and antitoxin. Toxin levels in recovered
food sample extracts were also detected and one MBA-negative sample was found to
contain 0.32 LD50 per ml extract. The FDC assay provides a real alternative for
public health laboratories to unambiguously confirm all cases of type A botulism
and, due to its sensitivity, a promising new tool in toxin pharmacokinetic
studies. Botulinum neurotoxins are one of the most potent toxins known to man. Current
methods of detection involve the quantification of the toxin but do not take
into account the percentage of the toxin that is active. At present the assay
used for monitoring the activity of the toxin is the mouse bioassay, which is
lengthy and has ethical issues due to the use of live animals. This report
demonstrates a novel assay that utilises the endopeptidase activity of the toxin
to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of
SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of
373 fg/mL and has been further developed into a high throughput method using a
microplate reader detecting down to 600 fg/mL of active toxin. The results show
clear differences between the toxin product and the placebo, which contains the
pharmaceutical excipients human serum albumin and lactose, showing that the
assay detects the active form of the toxin. |
Which factor interacts with Treslin/TICRR throughout the cell cycle of human cells? | MDM two binding protein (MTBP) is a factor that interacts with Treslin/TICRR throughout the cell cycle. MTBP depletion by means of small interfering RNA inhibits DNA replication by preventing assembly of the CMG (Cdc45-MCM-GINS) holohelicase during origin firing. Although MTBP has been implicated in the function of the p53 tumor suppressor, it is required for DNA replication irrespective of a cell's p53 status. MTBP is proposed to act with Treslin/TICRR to integrate signals from cell cycle and DNA damage response pathways to control the initiation of DNA replication in human cells. | |
What is oprozomib? | Oprozomib is a second-generation, highly-selective, orally administered proteasome inhibitor with promising activity against multiple myeloma.
Oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Consequently, oprozomib seems to be able to effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.
Oprozomib effectively decreases multiple myeloma cell viability.
Oprozomib potently inhibit cell survival and induce apoptosis in HNSCC cell lines via upregulation of pro-apoptotic Bik. Upregulation of Mcl-1 by these agents served to dampen their efficacies. Oprozomib also induced autophagy, mediated, in part, by activation of the UPR pathway involving upregulation of ATF4 transcription factor. Autophagy induction served a prosurvival role. Oral administration of ONX 0912 inhibited the growth of HNSCC xenograft tumors in a dose-dependent manner.
Oprozomib inhibited NF-κB expression. | Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone
therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting
pathologic bone destruction. In clinical trials, carfilzomib, a next generation
epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy
and decreased side effects compared with bortezomib. Carfilzomib and its orally
bioavailable analog oprozomib, effectively decreased MM cell viability following
continual or transient treatment mimicking in vivo pharmacokinetics.
Interactions between myeloma cells and the bone marrow (BM) microenvironment
augment the number and activity of bone-resorbing osteoclasts (OCs) while
inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth
and osteolytic lesions. At clinically relevant concentrations, carfilzomib and
oprozomib directly inhibited OC formation and bone resorption in vitro, while
enhancing osteogenic differentiation and matrix mineralization. Accordingly,
carfilzomib and oprozomib increased trabecular bone volume, decreased bone
resorption and enhanced bone formation in non-tumor bearing mice. Finally, in
mouse models of disseminated MM, the epoxyketone-based PIs decreased murine
5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data
demonstrate that, in addition to anti-myeloma properties, carfilzomib and
oprozomib effectively shift the bone microenvironment from a catabolic to an
anabolic state and, similar to bortezomib, may decrease skeletal complications
of MM. The proteasome inhibitor bortezomib has shown remarkable clinical success in the
treatment of multiple myeloma. However, the efficacy and mechanism of action of
bortezomib in solid tumor maligcies is less well understood. In addition, the
use of this first-in-class proteasome inhibitor is limited by several factors,
including off-target effects that lead to adverse toxicities. We recently
reported the impact and mechanisms of carfilzomib and oprozomib, second-in-class
proteasome inhibitors with higher specificities and reduced toxicities, against
head and neck squamous cell carcinoma (HNSCC). Carfilzomib and oprozomib
potently inhibit HNSCC cell survival and the growth of HNSCC tumors. Both
compounds promote upregulation of proapoptotic BIK and antiapoptotic MCL1, which
serves to mediate and attenuate, respectively, the killing activities of these
proteasome inhibitors. Both compounds also induce complete autophagic flux that
is partially dependent on activation of the unfolded protein response (UPR) and
upregulation of ATF4. Carfilzomib- and oprozomib-induced autophagy acts to
promote HNSCC cell survival. Our study indicates that the therapeutic benefit of
these promising proteasome inhibitors may be improved by inhibiting MCL1
expression or autophagy. The activity observed with proteasome inhibitors and immunomodulatory drugs
(IMIDs) in multiple myeloma (MM) has prompted the development of second- and
third-generation agents with similar, but not exactly the same, mechanisms of
action as their predecessors. This review summarizes the mechanism of action and
the available data on the clinical activity of novel proteasome inhibitors
(carfilzomib, oprozomib, ixazomib, and marizomib) and novel IMIDs
(pomalidomide), stressing the similarities and differences with bortezomib, and
with thalidomide and lenalidomide, respectively. In summary, these novel agents
have shown clinical activity as single agents and in combination with
dexamethasone, with similar or even higher efficacy than their parental drugs;
moreover, they may even overcome resistance, indicating that there are some
differences in their mechanisms of action and resistance. These data indicate
that both the inhibition of the proteasome and the modulation of the immune
system are good strategies to target MM tumor cells and this, along with the
absence of complete cross-resistance observed among these drugs, open new
avenues to optimize their use through the most appropriate sequencing and
combinations. Inhibition of proteasome, a proteolytic complex responsible for the degradation
of ubiquitinated proteins, has emerged as a powerful strategy for treatment of
multiple myeloma (MM), a plasma cell maligcy. First-in-class agent,
bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in
pre-clinical and in clinical studies. However, despite its high efficiency, a
large proportion of patients do not achieve sufficient clinical response.
Therefore, the development of a second-generation of proteasome inhibitors (PIs)
with improved pharmacological properties was needed. Recently, several of these
new agents have been introduced into clinics including carfilzomib, marizomib
and ixazomib. Further, new orally administered second-generation PI oprozomib is
being investigated. This review provides an overview of main mechanisms of
action of PIs in MM, focusing on the ongoing development and progress of novel
anti-proteasome therapeutics. Acquired resistance to proteasome inhibitors represents a considerable
impediment to their effective clinical application. Carfilzomib and its orally
bioavailable structural analog oprozomib are second-generation,
highly-selective, proteasome inhibitors. However, the mechanisms of acquired
resistance to carfilzomib and oprozomib are incompletely understood, and
effective strategies for overcoming this resistance are needed. Here, we
developed models of acquired resistance to carfilzomib in two head and neck
squamous cell carcinoma cell lines, UMSCC-1 and Cal33, through gradual exposure
to increasing drug concentrations. The resistant lines R-UMSCC-1 and R-Cal33
demonstrated 205- and 64-fold resistance, respectively, relative to the parental
lines. Similarly, a high level of cross-resistance to oprozomib, as well as
paclitaxel, was observed, whereas only moderate resistance to bortezomib (8- to
29-fold), and low level resistance to cisplatin (1.5- to 5-fold) was seen.
Synergistic induction of apoptosis signaling and cell death, and inhibition of
colony formation followed co-treatment of acquired resistance models with
carfilzomib and the histone deacetylase inhibitor (HDACi) vorinostat. Synergism
was also seen with other combinations, including oprozomib plus vorinostat, or
carfilzomib plus the HDACi entinostat. Synergism was accompanied by upregulation
of proapoptotic Bik, and suppression of Bik attenuated the synergy. The acquired
resistance models also exhibited elevated levels of MDR-1/P-gp. Inhibition of
MDR-1/P-gp with reversin 121 partially overcame carfilzomib resistance in
R-UMSCC-1 and R-Cal33 cells. Collectively, these studies indicate that combining
carfilzomib or oprozomib with HDAC or MDR-1/P-gp inhibitors may be a useful
strategy for overcoming acquired resistance to these proteasome inhibitors. |
Is there a relationship between junctin and ryanodine receptors? | Yes, junctin binds to ryanodine receptors within the junctional sarcoplasmic reticulum of calcium release units, and normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. | Junctin is a 26 kDa membrane protein that binds to calsequestrin, triadin, and
ryanodine receptors (RyRs) within the junctional sarcoplasmic reticulum of
calcium release units. The sequence of junctin includes a short N-terminal
cytoplasmic domain a single transmembrane domain, and a highly charged
C-terminal domain located in the sarcoplasmic reticulum lumen. Dog and mouse
junctins are highly conserved at the transmembrane domains, but the luminal
domains are more divergent. To probe the contribution of junctin to the
architecture of calcium release units in heart, we engineered transgenic mice
overexpressing canine junctin and examined the left ventricular myocardium by
electron microscopy. Overall architecture of calcium release units is similar in
control myocardium and in myocardium overexpressing junctin by 5-10-fold. In
both myocardia, junctional SR cisternae are closely associated with exterior
membranes (plasmalemma and transverse tubules). The cisternae are flat; they
contain a string of calsequestrin beads and are lined by a row of feet, or RyRs,
on the side facing the exterior membranes. T tubule surface density, measured as
the perimeter of T tubule profiles v area of section, is the same in transgenic
and control myocardia (305 v 289 nm/nm(2)). Three changes affecting the
junctional SR architecture are apparent in the myocardium overexpressing
junctin. One is a more tightly zippered appearance of the junctional SR
cisternae. The width of the junctional SR is narrower and less variable in
overexpressing than in control myocardium and the calsequestrin content is more
compact. A second change is the extension of zippered junctional SR domains to
non-junctional regions, which we term "frustrated" junctional SR. A third change
is an increase in the extent of association between SR and T tubules. In junctin
overexpressing myocardium junctional SR cisternae cover approximately 45% of the
surface of all T tubule profiles, while in control myocardium the coverage
approximately 30%. Junctional associations between SR and T tubules are
increased in size. We conclude that the increase in junctin expression affects
the packing of calsequestrin in the junctional SR and facilitates the
association of SR and T tubules. The junctional domain of sarcoplasmic reticulum (jSR) is specialized for
receiving signals from the plasmalemma-transverse tubules and for releasing Ca2+
during muscle activation. The junctional face of the jSR, facing the transverse
tubules, is occupied by a molecular complex composed of the transmembrane Ca2+
release channels (ryanodine receptors); the luminal protein calsequestrin (CSQ);
the 2 membrane proteins, junctin (Jct), and triadin (Tr), which mediate
CSQ-ryanodine receptor interactions; and several other components. Under the
conditions prevailing within the sarcoplasmic reticulum lumen (physiological
ionic strength, mostly due to K+ and Ca2+ ions), CSQ forms long linear polymers
and the fixed protein gel is clearly visible in the electron microscope. The
luminal domains of Jct and Tr are detectable but, overall, the 2 molecules are
not clearly delineated. Cardiac muscles either overexpressing or bearing null
mutations for 3 proteins of the junctional complex (CSQ, Jct, and Tr) reveal the
contribution of these 3 components to the general architecture of the jSR. Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary
complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the
junctional SR membrane. The physiological role for junctin in the luminal
regulation of RyR Ca(2+) release remains unresolved, but it appears to be
essential for proper cardiac function since ablation of junctin results in
increased ventricular automaticity. Given that the junctin levels are severely
reduced in human failing hearts, we performed an in-depth study of the
mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient
cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display
increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)]
([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+)
load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated
[Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR)
reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release,
evidenced by the development of spontaneous aftercontractions. Single channel
recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of
junctin produces a dual effect on the normally linear response of RyRs to
luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR
channels are less responsive to luminal [Ca(2+)]; conversely, high luminal
[Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus,
junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the
luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates
that junctin normally acts as an activator of RyR channels at low luminal
[Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover
occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is
possible that the activator-inhibitor role of junctin may be exerted under
periods of prevalent parasympathetic and sympathetic activity, respectively. RATIONALE: In cardiac dyads, junctional Ca2+ directly controls the gating of the
ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release
from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+
signals because of probe diffusion, so a junction-targeted Ca2+ sensor should
reveal new information on cardiac excitation-contraction coupling and its
modification in disease states.
OBJECTIVE: To investigate Ca2+ signaling in the oscopic space of cardiac
dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional
space.
METHODS AND RESULTS: By fusing GCaMP6f to the N terminus of triadin 1 or
junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it
colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer.
This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native
GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ osparks)
that were ≈50× smaller in volume than conventional Ca2+ sparks (measured with
diffusible indicators). The presence of the biosensor did not disrupt normal
Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and
timing of release measurements were improved, despite the small recording
volume. We could also visualize coactivation of subclusters of RyRs within a
single junctional region, as well as quarky Ca2+ release events.
CONCLUSIONS: This new, targeted biosensor allows selective visualization and
measurement of odomain Ca2+ dynamics in intact cells and can be used to give
mechanistic insights into dyad RyR operation in health and in disease states
such as when RyRs become orphaned. |
Which medication should be administered when managing patients with suspected acute opioid overdose? | Naloxone is opioid anagonist that should be administered for all patients with suspected acute opioid overdose. Intravenous naltrexone hydrochloride is usually administered, however, other formulations, including enteral methylnaltrexone, nebulized naloxone and subcutaneous naloxone, are under investigation and can be used under certain circumstances. | Drug and alcohol abuse continue to be commonly encountered problems in most
patient populations. To deal effectively with these problems, the primary care
physician must have a thorough knowledge of the pharmacology of commonly abused
drugs and the adjunctive agents used in treatment. Management of alcoholism may
involve a range of medical interventions, including the treatment of alcohol
intoxication, the use of benzodiazepines for alcohol withdrawal, and possibly
the short-term administration of disulfiram to maintain sobriety. Successful
management of cocaine or amphetamine abuse requires an understanding of the
powerful reinforcing properties of these drugs and the unique problems that
arise in the recovery period. Barbiturate intoxication and withdrawal are
potentially life-threatening events requiring skilled in-patient treatment.
Prolonged use of benzodiazepines can lead to drug dependence; successful
withdrawal involves gradual dosage reduction. Acute intoxication from marijuana
or hallucinogenic drugs may occasionally result in adverse reactions requiring
medical intervention, but significant withdrawal reactions are rare. Management
of opioid overdose, whether illicit or iatrogenic, requires the prompt and
skillful use of opioid overdose, whether illicit or iatrogenic, requires the
prompt and skillful use of opioid antagonists. Promising new pharmacologic
approaches are now being successfully applied to the management of opioid
dependence. An acceptance of nicotine as the addictive component of tobacco
smoke has led to the development of nicotine gum as substitution therapy for
cigarette smoking. Successful pharmacologic management of overdose or withdrawal
is often the prerequisite for effective long-term treatment and recovery. OBJECTIVE: To develop a clinical prediction rule to identify patients who can be
safely discharged one hour after the administration of naloxone for presumed
opioid overdose.
METHODS: Patients who received naloxone for known or presumed opioid overdose
were formally evaluated one hour later for multiple potential predictor
variables. Patients were classified into two groups: those with adverse events
within 24 hours and those without. Using classification and regression tree
methodology, a decision rule was developed to predict safe discharge.
RESULTS: Clinical findings from 573 patients allowed us to develop a clinical
prediction rule with a sensitivity of 99% (95% CI = 96% to 100%) and a
specificity of 40% (95% CI = 36% to 45%). Patients with presumed opioid overdose
can be safely discharged one hour after naloxone administration if they: 1) can
mobilize as usual; 2) have oxygen saturation on room air of >92%; 3) have a
respiratory rate >10 breaths/min and <20 breaths/min; 4) have a temperature of
>35.0 degrees C and <37.5 degrees C; 5) have a heart rate >50 beats/min and <100
beats/min; and 6) have a Glasgow Coma Scale score of 15.
CONCLUSIONS: This prediction rule for safe early discharge of patients with
presumed opioid overdose performs well in this derivation set but requires
validation followed by confirmation of safe implementation. The objective of this guideline is to assist poison center personnel in the
appropriate out-of-hospital triage and initial out-of-hospital management of
patients with a suspected ingestion of dextromethorphan by 1) describing the
process by which an ingestion of dextromethorphan might be managed, 2)
identifying the key decision elements in managing cases of dextromethorphan
ingestion, 3) providing clear and practical recommendations that reflect the
current state of knowledge, and 4) identifying needs for research. This
guideline applies to the ingestion of dextromethorphan alone. Co-ingestion of
additional substances could require different referral and management
recommendations depending on the combined toxicities of the substances. This
guideline is based on an assessment of current scientific and clinical
information. The expert consensus panel recognizes that specific patient care
decisions might be at variance with this guideline and are the prerogative of
the patient and the health professionals providing care, considering all of the
circumstances involved. This guideline does not substitute for clinical
judgment. The grade of recommendation is in parentheses. 1) All patients with
suicidal intent, intentional abuse, or in cases in which a malicious intent is
suspected (e.g., child abuse or neglect) should be referred to an emergency
department (Grade D). 2) Patients who exhibit more than mild effects (e.g.,
infrequent vomiting or somnolence [lightly sedated and arousable with speaking
voice or light touch]) after an acute dextromethorphan ingestion should be
referred to an emergency department (Grade C). 3) Patients who have ingested
5-7.5 mg/kg should receive poison center-initiated follow-up approximately every
2 hours for up to 4 hours after ingestion. Refer to an emergency department if
more than mild symptoms develop (Grade D). 4) Patients who have ingested more
than 7.5 mg/kg should be referred to an emergency department for evaluation
(Grade C). 5) If the patient is taking other medications likely to interact with
dextromethorphan and cause serotonin syndrome, such as monoamine oxidase
inhibitors or selective serotonin reuptake inhibitors, poison center-initiated
follow-up every 2 hours for 8 hours is recommended (Grade D). 6) Patients who
are asymptomatic and more than 4 hours have elapsed since the time of ingestion
can be observed at home (Grade C). 7) Do not induce emesis (Grade D). 8) Do not
use activated charcoal at home. Activated charcoal can be administered to
asymptomatic patients who have ingested overdoses of dextromethorphan within the
preceding hour. Its administration, if available, should only be carried out by
health professionals and only if no contraindications are present. Do not delay
transportation in order to administer activated charcoal (Grade D). 9) For
patients who have ingested dextromethorphan and are sedated or comatose,
naloxone, in the usual doses for treatment of opioid overdose, can be considered
for prehospital administration, particularly if the patient has respiratory
depression (Grade C). 10) Use intravenous benzodiazepines for seizures and
benzodiazepines and external cooling measures for hyperthermia (>104 degrees F,
>40 degrees C) for serotonin syndrome. This should be done in consultation with
and authorized by EMS medical direction, by a written treatment protocol or
policy, or with direct medical oversight (Grade C). 11) Carefully ascertain by
history whether other drugs, such as acetaminophen, were involved in the
incident and assess the risk for toxicity or for a drug interaction. BACKGROUND: Opioid overdose has a high mortality, but is often reversible with
appropriate overdose management and naloxone (opioid antagonist). Training in
these skills has been successfully trialled internationally with opioid users
themselves. Healthcare professionals working in substance misuse are in a prime
position to deliver overdose prevention training to drug users and may
themselves witness opioid overdoses. The best method of training dissemination
has not been identified. The study assessed post-training change in clinician
knowledge for managing an opioid overdose and administering naloxone, evaluated
the 'cascade method' for disseminating training, and identified barriers to
implementation.
METHODS: A repeated-measures design evaluated knowledge pre-and-post training. A
sub-set of clinicians were interviewed to identify barriers to implementation.
Clinicians from addiction services across England received training.
Participants self-completed a structured questionnaire recording overdose
knowledge, confidence and barriers to implementation.
RESULTS: One hundred clinicians were trained initially, who trained a further
119 clinicians (n=219) and thereafter trained 239 drug users. The mean composite
score for opioid overdose risk signs and actions to be taken was 18.3/26 (±3.8)
which increased to 21.2/26 (±4.1) after training, demonstrating a significant
improvement in knowledge (Z=9.2, p<0.001). The proportion of clinicians willing
to use naloxone in an opioid overdose rose from 77% to 99% after training.
Barriers to implementing training were clinician time and confidence, service
resources, client willingness and naloxone formulation.
CONCLUSIONS: Training clinicians how to manage an opioid overdose and administer
naloxone was effective. However the 'cascade method' was only modestly
successful for disseminating training to a large clinician workforce, with a
range of clinician and service perceived obstacles. Drug policy changes and
improvements to educational programmes for drug services would be important to
ensure successful implementation of overdose training internationally. |
In which nuclear compartments is heterochromatin located? | This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modification | The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA)
and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines.
Global histone hyperacetylation, leading to decondensation of interphase
chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation
and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein,
HP1, were reduced after HDAC inhibition. The observed changes in the protein
levels were accompanied by changes in their interphase patterns. In control
cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to
a thin layer at the nuclear periphery, whereas TSA and NaBt caused the
peripheral regions to become intensely acetylated at H3(K9) and dimethylated at
H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the
nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1
proteins were repositioned more internally in the nucleus, being closely
associated with interchromatin compartments, while centromeric heterochromatin
was relocated closer to the nuclear periphery. These findings strongly suggest
dissociation of HP1 proteins from peripherally located centromeres in a
hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition
of histone deacetylases caused dynamic reorganization of chromatin in parallel
with changes in its epigenetic modifications. Ty3/gypsy elements represent one of the most abundant and diverse
LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their
evolutionary dynamics have not been explored in detail. Here, we conduct an in
silico analysis of the distribution and abundance of the full complement of 1045
copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy
elements is inversely related to arm length, with densities being greatest on
the X, and greater on the short versus long arms of both autosomes. Taking into
account the different heterochromatic and euchromatic compartments of the
genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along
each chromosome arm is determined mainly by the different proportions of
heterochromatin, particularly pericentric heterochromatin, relative to total arm
length. Additionally, the breakpoint regions of chromosomal inversion 2La
appears to be a haven for LTRrs. These elements are underrepresented more than
7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with
genes. The euchromatin on chromosome 3R shows a faster turnover rate of
Ty3/gypsy elements, characterized by a deficit of proviral sequences and the
lowest average sequence divergence of any autosomal region analyzed in this
study. This probably reflects a principal role of purifying selection against
insertion for the preservation of longer conserved syntenyc blocks with adaptive
importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent
activity, an important fraction are inactive remts of relatively ancient
insertions apparently subject to genetic drift. Consistent with these
computational predictions, an analysis of the occupancy rate of putatively older
insertions in natural populations suggested that the degenerate copies have been
fixed across the species range in this mosquito, and also are shared with the
sibling species Anopheles arabiensis. |
Have mutations in the ZEB2 gene been found in any human syndrome? | Yes, the Mowat-Wilson syndrome | MWS is a multiple congenital anomaly syndrome, first clinically delineated by
Mowat et al in 1998. Over 45 cases have now been reported. All patients have
typical dysmorphic features in association with severe intellectual disability,
and nearly all have microcephaly and seizures. Congenital anomalies, including
Hirschsprung disease (HSCR), congenital heart disease, hypospadias,
genitourinary anomalies, agenesis of the corpus callosum, and short stature are
common. The syndrome is the result of heterozygous deletions or truncating
mutations of the ZFHX1B (SIP1) gene on chromosome 2q22. We report a girl who had Hirschsprung disease in association with distinct
facial appearance, microcephaly, agenesis of the corpus callosum and mental
retardation (Mowat-Wilson syndrome). Mutation analysis of the zinc finger homeo
box 1 B (ZFHX1 B) gene revealed a de novo 7 bp deletion (TGGCCCC) at nucleotide
1773 (1773 delTGGCCCC) resulting in a frameshift and leading to a termination
codon at amino acid residue 604 (604 X) in exon 8 C. The zinc finger homeo box 1
B (Smad interacting protein-1) is a transcription corepressor of Smad target
genes with functions in the patterning of neural crest derived cells, CNS, and
midline structures. Mutations in ZFHX1 B can lead to neurological disorders in
addition to dysmorphic features, megacolon, and other malformations. ZFHX1B encodes Smad-interacting protein 1, a transcriptional corepressor
involved in the transforming growth factors beta (TGFbeta) signaling pathway.
ZFHX1B mutations cause a complex developmental phenotype characterized by severe
mental retardation (MR) and multiple congenital defects. We compared the
distribution of ZFHX1B transcripts during mouse and human embryogenesis as well
as in adult mice and humans. This showed that this gene is strongly transcribed
at an early stage in the developing peripheral and central nervous systems of
both mice and humans, in all neuronal regions of the brains of 25-week human
fetuses and adult mice, and at varying levels in numerous nonneural tissues.
Northern blot analysis suggested that ZFHX1B undergoes tissue-specific
alternative splicing in both species. These results strongly suggest that ZFHX1B
determines the transcriptional levels of target genes in various tissues through
the combinatorial interactions of its isoforms with different Smad proteins.
Thus, as well as causing neural defects, ZFHX1B mutations may also cause other
malformations. Hypospadias, when the urethra opens on the ventral side of the penis, is a
common malformation seen in about 3 per 1,000 male births. It is a complex
disorder associated with genetic and environmental factors and can be part of
genetic syndromes. Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly
syndrome characterized by a distinct facial phenotype, Hirschsprung disease,
microcephaly and mental retardation. It is caused by mutations in the zinc
finger homeo box 1B gene, ZFHX1B (SIP1). To date, 68 deletion/mutation-positive
cases have been reported. Genitourinary anomalies are common in MWS. Here we
report that hypospadias is common in males with this syndrome. In 39 patients
where this information was available, hypospadias was present in 46% of patients
(18/39). In the 3 Italian male cases reported here, hypospadias was always
present. MWS should be considered by endocrinologists in patients with
hypospadias associated with developmental delays/mental retardation, in
particular in the presence of a distinct facial phenotype. Mowat-Wilson Syndrome is a recently delineated mental retardation syndrome
usually associated with multiple malformations and a recognizable facial
phenotype caused by defects of the transcriptional repressor ZFHX1B. To address
the question of clinical and mutational variability, we analysed a large number
of patients with suspected Mowat-Wilson Syndrome (MWS). Without prior knowledge
of their mutational status, 70 patients were classified into "typical MWS",
"ambiguous" and "atypical" groups according to their facial phenotype. Using
FISH, qPCR and sequencing, ZFHX1B deletions, splice site or truncating mutations
were detected in all 28 patients classified as typical MWS. No ZFHX1B defect was
apparent in the remaining 15 cases with ambiguous facial features or in the 27
atypical patients. Genotype-phenotype analysis confirmed that ZFHX1B deletions
and stop mutations result in a recognizable facial dysmorphism with associated
severe mental retardation and variable malformations such as Hirschsprung
disease and congenital heart defects. Our findings indicate that structural eye
anomalies such as microphthalmia should be considered as part of the MWS
spectrum. We also show that agenesis of the corpus callosum and urogenital
anomalies (especially hypospadias) are significant positive predictors of a
ZFHX1B defect. Based on our observation of affected siblings and the number of
MWS cases previously reported, we suggest a recurrence risk of around 1%. The
lack of missense mutations in MWS and MWS-like patients suggests there may be
other, as yet unrecognized phenotypes, associated with missense mutations of
this transcription factor. BACKGROUND/PURPOSE: Patients with zinc finger homeo box 1B (ZFHX1B) mutations or
deletions develop multiple congenital anomalies including Hirschsprung disease,
known as Mowat-Wilson syndrome (MWS). In this study, we investigated variations
in the enteric neural plexus abnormalities in MWS using morphometry-based
histopathologic analysis.
METHODS: Seven patients with MWS (3 with mutations in exon 8 of ZFHX1B and 4
with deletions) who had undergone modified Duhamel's operations for Hirschsprung
disease were examined. Surgically resected rectosigmoid specimens were analyzed
morphometrically.
RESULTS: The length of the aganglionic segment was longer than 3 cm in all the
patients with deletions. In 3 patients with mutations, the aganglionic region
was not detected in the surgically resected specimens; however, the parameters
of the ganglions and plexus were significantly smaller than those of controls
(cloaca and aproctia), indicative of a transitional zone. Variation in the
severity of pathological changes among the 3 patients with mutations was also
noted.
CONCLUSIONS: The variations in myenteric plexus pathologies in MWS appear to be
caused by both variations in ZFHX1B abnormalities and epigenetic factors. Mowat-Wilson syndrome is a recently delineated severe mental retardation,
multiple congenital anomalies syndrome caused by domit nonsense or frameshift
mutations, deletions or translocations of the zinc finger homeobox 1B gene
(ZFHX1B). We report on a patient with exceptional mild phenotype caused by a
novel and unusual splice mutation in the 5'UTR. The aberrant transcript leads to
usage of an alternative upstream start codon. The resulting protein differs from
the wild-type only in the first 24 amino acids. The aberrant protein therefore
contains all known functional domains, but might lack a so far unrecognized
putative N-terminal acylation site, which is probably important for neuronal
function and facial structures. Mowat-Wilson syndrome (MWS) is a rare mental retardation-multiple congenital
anomalies syndrome associated with typical facial dysmorphism. Patients can show
a variety of other anomalies like short stature, microcephaly, Hirschsprung
disease, malformations of the brain, seizures, congenital heart defects and
urogenital anomalies. Mutations leading to haploinsufficiency of the ZFHX1B gene
have been described as the underlying cause of this condition. We report on the
clinical findings in a 2(1/2)-year-old boy with some aspects out of the
MWS-spectrum in addition to unusual anomalies and a novel missense mutation in
the ZFHX1B gene. Mowat-Wilson syndrome (MWS) is a recently delineated mental retardation
(MR)-multiple congenital anomaly syndrome, characterized by typical facies,
severe MR, epilepsy, and variable congenital malformations, including
Hirschsprung disease (HSCR), genital anomalies, congenital heart disease (CHD),
and agenesis of the corpus callosum (ACC). It is caused by de novo heterozygous
mutations or deletions of the ZFHX1B gene located at 2q22. ZFHX1B encodes
Smad-interacting protein-1 (SMADIP1 or SIP1), a transcriptional corepressor
involved in the transforming growth factor-beta signaling pathway. It is a
highly evolutionarily conserved gene, widely expressed in embryological
development. Over 100 mutations have been described in patients with clinically
typical MWS, who almost always have whole gene deletions or truncating mutations
(nonsense or frameshift) of ZFHX1B, suggesting that haploinsufficiency is the
basis of MWS pathology. No obvious genotype-phenotype correlation could be
identified so far, but atypical phenotypes have been reported with missense or
splice mutations in the ZFHX1B gene. In this work we describe 40 novel mutations
and we summarize the various mutational reports published since the
identification of the causative gene. High-resolution analyses of complex chromosome rearrangements (CCR) have
demonstrated in individuals with abnormal phenotypes that not all seemingly
balanced CCRs based on G-banding are completely balanced at breakpoint level.
Here we report on an apparently balanced de novo CCR involving chromosomes 2, 3
and 5 present in a 6-month-old girl. She was referred for genetic evaluation
because of severe psychomotor retardation, distinctive dysmorphic features and
microcephaly. A 1Mb resolution array-CGH analysis of DNA from the patient
revealed a deletion of about 6Mb for chromosome 2. FISH analysis showed that the
deletion interval found in band 2q22 mapped at the translocation breakpoint, and
that the ZFHX1B gene, which is known to be involved in the Mowat-Wilson
syndrome, is located within the deletion interval. To our knowledge this is the
first case of a complex chromosomal rearrangement associated with Mowat-Wilson
syndrome. Our data illustrate the important role for high-resolution
investigation of apparently balanced chromosome rearrangements in patients with
unexplained psychomotor retardation and/or other clinical features, and should
contribute to our understanding of the mechanisms involved in chromosome
rearrangement. Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome
characterized by a distinct facial phenotype (high forehead, frontal bossing,
large eyebrows, medially flaring and sparse in the middle part, hypertelorism,
deep set but large eyes, large and uplifted ear lobes, with a central
depression, saddle nose with prominent rounded nasal tip, prominent columella,
open mouth, with M-shaped upper lip, frequent smiling, and a prominent but
narrow and triangular pointed chin), moderate-to-severe intellectual deficiency,
epilepsy and variable congenital malformations including Hirschsprung disease
(HSCR), genitourinary anomalies (in particular hypospadias in males), congenital
heart defects, agenesis of the corpus callosum and eye anomalies. The prevalence
of MWS is currently unknown, but 171 patients have been reported so far. It
seems probable that MWS is under-diagnosed, particularly in patients without
HSCR. MWS is caused by heterozygous mutations or deletions in the Zinc finger
E-box-binding homeobox 2 gene, ZEB2, previously called ZFHX1B (SIP1). To date,
over 100 deletions/mutations have been reported in patients with a typical
phenotype; they are frequently whole gene deletions or truncating mutations,
suggesting that haploinsufficiency is the main pathological mechanism. Studies
of genotype-phenotype analysis show that facial gestalt and delayed psychomotor
development are constant clinical features, while the frequent and severe
congenital malformations are variable. In a small number of patients, unusual
mutations can lead to an atypical phenotype. The facial phenotype is
particularly important for the initial clinical diagnosis and provides the
hallmark warranting ZEB2 mutational analysis, even in the absence of HSCR. The
majority of MWS cases reported so far were sporadic, therefore the recurrence
risk is low. Nevertheless, rare cases of sibling recurrence have been observed.
Congenital malformations and seizures require precocious clinical investigation
with intervention of several specialists (including neonatologists and
pediatricians). Psychomotor development is delayed in all patients, therefore
rehabilitation (physical therapy, psychomotor and speech therapy) should be
started as soon as possible. We herein report 3 cases of Mowat-Wilson syndrome, characterized by distinct
facial features, severe psychomotor retardation, and epilepsy, recurring in 3
siblings from the same parents. The proband was a 15-month-old boy, the youngest
of 3 children (2 elder sisters), who was referred to our hospital for the
treatment of severe seizures. The clinical features and course of these 3
siblings were compatible with those of previously reported Mowat-Wilson syndrome
patients, and all siblings had the same E87X nonsense mutation in ZFHX1B,
whereas their mother did not show the mutation. Because Mowat-Wilson syndrome
has been caused by de novo mutation in ZFHX1B, germ-line mosaicism should be
considered if recurrence in siblings is observed. INTRODUCTION: Mowat-Wilson syndrome is a congenital syndrome caused by a defect
of the transcriptional repressor ZFHX1B (SIP1) gene on the chromosome 2q22-q23.
The genotype-phenotype analysis confirmed that ZFHX1B deletions and mutations
result in a recognizable facial dysmorphism with a multiple congenital anomaly
and mental retardation.
CASE REPORT: This report is about one new patient from Croatia with the typical
phenotype. Molecular genetic studies showed the novel mutation in ZFHX1B (exon
8: c.2372del C; p.T791fsX816). This mutation has not been reported before. The
literature is reviewed.
CONCLUSION: Mowat-Wilson syndrome is a newly described congenital syndrome and
should be considered in any individual with characteristic facial features and
mental retardation in associations with congenital malformations. Mowat-Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by
heterozygous mutations or deletions of the ZEB2 gene, and characterized by
typical face, moderate-to-severe mental retardation, epilepsy, Hirschsprung
disease, and multiple congenital anomalies, including genital anomalies
(particularly hypospadias in males), congenital heart defects, agenesis of the
corpus callosum, and eye defects. Since the first delineation by Mowat et al.
[Mowat et al. (1998); J Med Genet 35:617-623], approximately 179 patients with
ZEB2 mutations, deletions or cytogenetic abnormalities have been reported
primarily from Europe, Australia and the United States. Genetic defects include
chromosome 2q21-q23 microdeletions (or different chromosome rearrangements) in
few patients, and ZEB2 mutations in most. We report on clinical and genetic data
from 19 Italian patients, diagnosed within the last 5 years, including six
previously published, and compare them with patients already reported. The main
purpose of this review is to underline a highly consistent phenotype and to
highlight the phenotypic evolution occurring with age, particularly of the
facial characteristics. The prevalence of MWS is likely to be underestimated.
Knowledge of the phenotypic spectrum of MWS and of its changing phenotype with
age can improve the detection rate of this condition. Mowat-Wilson syndrome is a genetic condition characterized by a recognizable
facial phenotype in addition to moderate to severe cognitive disability with
severe speech impairment and variable multiple congenital anomalies. The
anomalies may include Hirschsprung disease, heart defects, structural eye
anomalies including microphthalmia, agenesis of the corpus callosum, and
urogenital anomalies. Microcephaly, seizure disorder and constipation are
common. All typical cases result from haploinsufficiency of the ZEB2 (also known
as ZFHX1B or SIP-1) gene, with over 100 distinct mutations now described.
Approximately 80% of patients have a nonsense or frameshift mutation detectable
by sequencing, with the rest having gross deletions necessitating a dosage
sensitive assay. Here we report on the results of comprehensive molecular
testing for 27 patients testing positive for MWS. Twenty-one patients had a
nonsense, frameshift, or splice site mutation identified by sequencing; 14 of
which localized to exon 8 and 17 of which are novel. Six patients had deletions
in the ZEB2 gene, including two novel partial gene deletions. This report, the
first such analysis in North American patients, adds to the growing list of both
novel pathogenic mutations associated with MWS, as well as other variants in the
ZEB2 gene. In addition, we suggest an economical testing strategy. BACKGROUND: Fetuses with increased nuchal translucency but apparently normal
karyotypes may have small genetic defects that are undetectable by conventional
cytogenetic studies. Microarray comparative genomic hybridization (array
comparative genomic hybridization) may help prenatal diagnosis by revealing
small genetic defects.
CASE: A patient presented with a fetus with large nuchal translucency and
ambiguous genitalia at 13 weeks of gestation. Conventional fetal karyotype by
chorionic villus sampling was 46,XY,inv (1)(p31q42). The inversion was de novo.
Further analysis by array comparative genomic hybridization revealed a
single-copy ZEB2 gene deletion at 2q22.3 consistent with Mowat-Wilson syndrome.
Ultrasonography at 17 weeks revealed a reduced nuchal fold of 5 mm. The patient
decided to terminate the pregcy, which was completed uneventfully at 17 weeks
of gestation.
CONCLUSION: Array comparative genomic hybridization is a useful complementary
diagnostic tool in fetuses with increased nuchal translucency but apparently
normal karyotypes. Rubinstein-Taybi syndrome (RSTS) is a rare autosomal domit disorder
characterised by facial dysmorphisms, growth and psychomotor development delay,
and skeletal defects. The known genetic causes are point mutations or deletions
of the CREBBP (50-60%) and EP300 (5%) genes. To detect chromosomal
rearrangements indicating novel positional candidate RSTS genes, we used a-CGH
to study 26 patients fulfilling the diagnostic criteria for RSTS who were
negative at fluorescence in situ hybridisation analyses of the CREBBP and EP300
regions, and direct sequencing analyses of the CREBBP gene. We found seven
imbalances (27%): four de novo and three inherited rearrangements not reported
among the copy number variants. A de novo 7p21.1 deletion of 500 kb included the
TWIST1 gene, a suggested candidate for RSTS that is responsible for the
Saethre-Chotzen syndrome, an entity that enters in differential diagnosis with
RSTS. A similar issue of differential diagnosis was raised by a large 4.3 Mb
2q22.3q23.1 deletion encompassing ZEB2, the gene responsible for the
Mowat-Wilson syndrome, whose signs may overlap with RSTS. Positional candidate
genes could not be sought in the remaining pathogenetic imbalances, because of
the size of the involved region (a 9 Mb 2q24.3q31.1 deletion) and/or the
relative paucity of suitable genes (a 5 Mb 3p13p12.3 duplication). One of the
inherited rearrangements, the 17q11.2 379Kb duplication, represents the
reciprocal event of the deletion underlying an overgrowth syndrome, both being
mediated by the NF1-REP-P1 and REP-P2 sub-duplicons. The contribution of this
and the other detected CNVs to the clinical RSTS phenotype is difficult to
assess. We present a clinical case of a female infant with multiple anomalies and
distinctive facial features, with an exceptionally severe clinical course of
Hirschsprung disease. The girl was also diagnosed with Mowat-Wilson syndrome,
confirmed by molecular analysis as a heterozygous deletion of the ZEB2 gene.
Moreover, molecular karyotyping revealed a deletion involving further genes
(KYNU, ARHGAP15, and GTDC1). Mowat-Wilson syndrome (MWS) is an autosomal domit intellectual disability
syndrome characterised by unique facial features and congenital anomalies such
as Hirschsprung disease, congenital heart defects, corpus callosum agenesis and
urinary tract anomalies. Some cases also present epilepsy, growth retardation
and microcephaly. The syndrome is caused by mutations or deletions of the ZEB2
gene at chromosome 2q22-q23. MWS was first described in 1998 and until now
approximately 180 cases have been reported worldwide. We report the first three
molecularly confirmed Danish cases with MWS. Mowat-Wilson syndrome is a genetic disorder characterized by a distinct facial
appearance, moderate-to-severe mental retardation, microcephaly, agenesis of the
corpus callosum, Hirschsprung disease, congenital heart disease, and genital
anomalies. Ophthalmological abnormalities have been rarely described in patients
with this condition which is caused by mutations in the ZEB2 gene. We report a
9-year-old female with this syndrome who has severe ocular abnormalities
including bilateral microphthalmia, cataract, and retinal aplasia. The zinc-finger, E-box-binding homeobox-2 (Zeb2) gene encodes a SMAD-interacting
transcription factor that has diverse roles in development and disease.
Mutations at the hZeb2 locus cause Mowat-Wilson syndrome (MWS), a genetic
disorder that is associated with mental retardation and other, case- and
sex-dependent clinical features. Recent studies have detailed microRNA-mediated
control of Zeb2, but little is known about the genomic context of this gene or
of enhancer sequences that may direct its diverse functions. Here, we describe a
novel transgenic rodent model in which Zeb2 regulatory sequence has been
disrupted, resulting in a postnatal developmental phenotype that is autosomal
domit. The phenotype exhibits a genotype-by-sex interaction and manifests
primarily as an acute attenuation of postnatal kidney development in males.
Other aspects of embryonic and neonatal development, including neuronal, are
unaffected. The transgene insertion site is associated with a 12 kb deletion,
1.2 Mb upstream of Zeb2, within a 4.1 Mb gene desert. A conserved sequence,
derived from the deleted region, enhanced Zeb2 promoter activity in
transcription assays. Tissue and temporal restriction of this enhancer activity
may involve postnatal changes in proteins that bind this sequence. A control
human/mouse VISTA enhancer (62 kb upstream of Zeb2) also up-regulated the Zeb2
promoter, providing evidence of a string of conserved distal enhancers. The
phenotype arising from deletion of one copy of the extreme long-range enhancer
indicates a critical role for this enhancer at one developmental stage.
Haploinsufficiency of Zeb2 in this developmental context reflects inheritance of
MWS and may underlie some sex-dependent, non-neural characteristics of this
human inherited disorder. Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder
occurring in the progeny of a single bull. Its clinical spectrum includes
polledness (complete agenesis of horns), facial dysmorphism, growth delay,
chronic diarrhea, premature ovarian failure, and variable neurological and
cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio,
suggesting male lethality during pregcy. Using Mendelian error mapping and
whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine
chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced
control and affected 90-day old fetuses to characterize this syndrome by
histological and expression analyses. Compared to wild type individuals,
affected animals showed a decreased expression of the three deleted genes. Based
on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of
ZEB2, is responsible for most of the effects of the mutation. Finally
sperm-FISH, embryo genotyping and analysis of reproduction records confirmed
somatic mosaicism in the founder bull and male-specific lethality during the
first third of gestation. In conclusion, we identified a novel locus involved in
bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition
plays a critical role in horn bud differentiation. We also provide new insights
into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and
describe the first case of male-specific lethality associated with an autosomal
locus in a non-murine mammalian species. This result sets PMS as a unique model
to study sex-specific gene expression/regulation. Mowat-Wilson syndrome (OMIM 235730) is a genetic condition characterized by
moderate-to-severe intellectual disability, a recognizable facial phenotype, and
multiple congenital anomalies. The striking facial phenotype in addition to
other features such as severely impaired speech, hypotonia, microcephaly, short
stature, seizures, corpus callosum agenesis, congenital heart defects,
hypospadias, and Hirschsprung disease are particularly important clues for the
initial clinical diagnosis. All molecularly confirmed cases with typical MWS
have a heterozygous loss-of-function mutation in the zinc finger E-box protein 2
(ZEB2) gene, also called SIP1 (Smad-interacting protein 1) and ZFHX1B,
suggesting that haploinsufficiency is the main pathological mechanism.
Approximately 80% of mutations are nonsense and frameshift mutations (small
insertions or deletions). About half of these mutations are located in exon
eight. Here, we report the first Indonesian patient with Mowat-Wilson syndrome
confirmed by molecular analysis. Mowat-Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive
facial gestalt-multiple congenital anomaly syndrome, commonly associating
microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects,
urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de
novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to
haplo-insufficiency through premature stop codons or large gene deletions. Only
three missense mutations have been reported so far; none of which resides in a
known functional domain of ZEB2. In this study, we report and analyze the
functional consequences of three novel missense mutations, p.Tyr1055Cys,
p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger
(C-ZF) domain of ZEB2. Patients' phenotype included the facial gestalt of MWS
and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies
showed that all the three mutations prevented binding and repression of the
E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the
zebrafish morphant technology, we performed rescue experiments using wild-type
(WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue,
correlating with the severity of patients' phenotype, was observed. Our data
provide evidence that these missense mutations cause a partial loss of function
of ZEB2, suggesting that its role is not restricted to repression of E-cadherin.
Functional domains other than C-ZF may play a role in early embryonic
development. Finally, these findings broaden the clinical spectrum of ZEB2
mutations, indicating that MWS ought to be considered in patients with lesser
degrees of ID and a suggestive facial gestalt, even in the absence of congenital
malformation. Mowat-Wilson syndrome (MWS) is a genetic disease caused by heterozygous
mutations or deletions of the ZEB2 gene rarely diagnosed prenatally and with
little fetal description reported. It is mainly characterized by
moderate-to-severe intellectual disability, epilepsy, facial dysmorphism and
various malformations including Hirschsprung disease and corpus callosum
anomalies. Here we report a fetal case of MWS well described, suspected at
standard autopsy. The association of a corpus callosum hypoplasia with a
histological Hirschsprung disease and a typical facial gestalt allowed the
guiding of genetic testing. Classical fetopathological examination still keeps
indications in cases of syndromic association in the era of virtual autopsy. Individuals with Mowat-Wilson syndrome (MWS; OMIM#235730) have characteristic
facial features, a variety of congenital anomalies such as Hirschsprung disease,
and intellectual disabilities caused by mutation or deletion of ZEB2 gene. This
deletion or cytogenetic abnormality has been reported primarily from Europe,
Australia and the United States, but not in Korea. Here we report a patient with
characteristic facial features of MWS, developmental delay and spasticity. High
resolution microarray analysis revealed 0.9 Mb deletion of 2q22.3 involving two
genes: ZEB2 and GTDC1. This case shows the important role of high resolution
microarray in patients with unexplained psychomotor retardation and/or facial
dysmorphism. Knowledge about the most striking clinical signs and implementation
of effective molecular tests like microarray could significantly increase the
detection rate of new cases of MWS in Korea. This is the first reported case of
MWS in Korea. Mowat-Wilson syndrome is a genetic disease caused by heterozygous mutations or
deletions of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. The syndrome
is characterized by typical facial features, moderate-to-severe mental
retardation, epilepsy and variable congenital malformations, including
Hirschsprung disease, genital anomalies, congenital heart disease, agenesis of
the corpus callosum, and eye defects. The prevalence of Mowat-Wilson syndrome is
currently unknown, but it seems that Mowat-Wilson syndrome is underdiagnosed,
particularly in patients without Hirschsprung disease. We report here the first
Egyptian case of Mowat-Wilson syndrome who was conceived by intracytoplasmic
sperm injection. The patient manifested bilateral sensorineural hearing loss--a
new feature not previously reported in cases of Mowat-Wilson syndrome. This
report describes the first Egyptian patient of Mowat-Wilson syndrome who was
conceived after intracytoplasmic sperm injection, and provides a new evidence
for the inclusion of deafness among the congenital defects of the syndrome. |
Which enzymes are involved in global genome nucleotide excision repair (GG-NER) in bacteria? | Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. Damage recognition during bacterial NER depends upon UvrA, which binds to the damage and loads UvrB onto the DNA. Subsequently, UvrA, UvrB and UvrC form the excinuclease protein UvrABC endonuclease, a multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. In some cases, Cho may be the effective nuclease for NER, rather than UvrC. UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. In gram-positive organisms, PcrA helicase can also displace UvrC and the excised oligonucleotide from a post-incision NER complex. | DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually
bulky nature. We have recently shown that nucleotide excision repair (NER) and
RecBCD-dependent homologous recombination (HR) collaboratively alleviate the
lethal effect of DPCs in Escherichia coli. In this study, to gain further
insight into the damage-processing mechanism for DPCs, we assessed the
sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing
agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that
the damage tolerance mechanism involving HR and subsequent replication restart
(RR) provides the most effective means of cell survival against DPCs.
Translesion synthesis does not serve as an alternative damage tolerance
mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies
primarily on NER, which provides a second and moderately effective means of cell
survival against DPCs. Interestingly, Cho rather than UvrC seems to be an
effective nuclease for the NER of DPCs. Together with the genes responsible for
HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair
and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly
but significantly sensitive to FA but not azaC, possibly reflecting the
complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional
role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on
cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the
repair of DPCs, presumably by removing 5-azacytosine or its degradation product
from the chromosome. Nucleotide excision repair (NER) is universally used to recognize and remove
many types of DNA damage. In eubacteria, the NER system typically consists of
UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition,
when DNA damage blocks transcription, transcription-repair coupling factor
(TRCF), the product of the mfd gene, recruits the Uvr complex to repair the
damage. Previous work using selected mutants and assays have indicated that
pathogenic Neisseria spp. carry a functional NER system. In order to
comprehensively examine the role of NER in Neisseria gonorrhoeae DNA
recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC,
uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and
uvrD mutants were complemented with a copy of each gene in an ectopic locus.
Each uvr mutant strain was highly sensitive to UV irradiation and also showed
sensitivity to hydrogen peroxide killing, confirming that all of the NER genes
in N. gonorrhoeae are functional. The effect of RecA expression on UV survival
was minor in uvr mutants but much larger in the mfd mutant. All of the NER
mutants demonstrated wild-type levels of pilin antigenic variation and DNA
transformation. However, the uvrD mutant exhibited higher frequencies of
PilC-mediated pilus phase variation and spontaneous mutation, a finding
consistent with a role for UvrD in mismatch repair. We conclude that NER
functions are conserved in N. gonorrhoeae and are important for the DNA repair
capabilities of this strict human pathogen. The nucleotide excision repair mechanism (NER) of Escherichia coli is
responsible for the recognition and elimination of more than twenty different
DNA lesions. Herein, we evaluated the in vivo role of NER in the repair of DNA
adducts generated by psoralens (mono- or bi-functional) and UV-A light (PUVA) in
E. coli. Cultures of wild-type E. coli K12 and mutants for uvrA, uvrB, uvrC or
uvrAC genes were treated with PUVA and cell survival was determined. In
parallel, kinetics of DNA repair was also evaluated by the comparison of DNA
sedimentation profiles in all the strains after PUVA treatment. The uvrB mutant
was more sensitive to PUVA treatment than all the other uvr mutant strains.
Wild-type strain, and uvrA and uvrC mutants were able to repair PUVA-induced
lesions, as seen by DNA sedimentation profiles, while the uvrB mutant was unable
to repair the lesions. In addition, a quadruple fpg nth xth nfo mutant was
unable to nick PUVA-treated DNA when the crude cell-free extract was used to
perform plasmid nicking. These data suggest that DNA repair of PUVA-induced
lesions may require base excision repair functions, despite proficient UvrABC
activity. These results point to a specific role for UvrB protein in the repair
of psoralen adducts, which appear to be independent of UvrA or UvrC proteins, as
described for the classical UvrABC endonuclease mechanism. Transcription-coupled DNA repair (TCR) is a subpathway of nucleotide excision
repair (NER) that is triggered when RNA polymerase is stalled by DNA damage.
Lesions targeted by TCR are repaired more quickly than lesions repaired by the
transcription-independent "global" NER pathway, but the mechanism underlying
this rate enhancement is not understood. Damage recognition during bacterial NER
depends upon UvrA, which binds to the damage and loads UvrB onto the DNA.
Bacterial TCR additionally requires the Mfd protein, a DNA translocase that
removes the stalled transcription complexes. We have determined the properties
of Mfd, UvrA, and UvrB that are required for the elevated rate of repair
observed during TCR. We show that TCR and global NER differ in their
requirements for damage recognition by UvrA, indicating that Mfd acts at the
very earliest stage of the repair process and extending the functional
similarities between TCR in bacteria and eukaryotes. BACKGROUND: Extensive genetic diversity and rapid allelic diversification are
characteristics of the human gastric pathogen Helicobacter pylori, and are
believed to contribute to its ability to cause chronic infections. Both a high
mutation rate and frequent imports of short fragments of exogenous DNA during
mixed infections play important roles in generating this allelic diversity. In
this study, we used a genetic approach to investigate the roles of nucleotide
excision repair (NER) pathway components in H. pylori mutation and
recombination.
RESULTS: Inactivation of any of the four uvr genes strongly increased the
susceptibility of H. pylori to DNA damage by ultraviolet light. Inactivation of
uvrA and uvrB significantly decreased mutation frequencies whereas only the uvrA
deficient mutant exhibited a significant decrease of the recombination frequency
after natural transformation. A uvrC mutant did not show significant changes in
mutation or recombination rates; however, inactivation of uvrC promoted the
incorporation of significantly longer fragments of donor DNA (2.2-fold increase)
into the recipient chromosome. A deletion of uvrD induced a
hyper-recombinational phenotype.
CONCLUSIONS: Our data suggest that the NER system has multiple functions in the
genetic diversification of H. pylori, by contributing to its high mutation rate,
and by controlling the incorporation of imported DNA fragments after natural
transformation. Cisplatin is currently used in tumor chemotherapy to induce the death of
maligt cells through blockage of DNA replication. It is a commonly used
chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA.
Escherichia coli K12 mutant strains deficient in nucleotide excision repair
(NER) were submitted to increasing concentrations of cisplatin, and the results
revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and
cho mutants remain as the wild type strain. The time required for both gene
expression turn-off and return to normal weight DNA in wild-type E. coli was not
accomplished even after 4 h post-treatment with cisplatin, while the same
process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a
heavily damaging action of cisplatin can be seen not only by persistent nicks on
genomic DNA, but also by NER gene expression exceeding manifold that seen after
equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB
gene expression from its putative upstream promoter P3 in an SOS-independent
manner. The incorporation of ribonucleotides in DNA has attracted considerable notice in
recent years, since the pool of ribonucleotides can exceed that of the
deoxyribonucleotides by at least 10-20-fold, and single ribonucleotide
incorporation by DNA polymerases appears to be a common event. Moreover
ribonucleotides are potentially mutagenic and lead to genome instability. As a
consequence, errantly incorporated ribonucleotides are rapidly repaired in a
process dependent upon RNase H enzymes. On the other hand, global genomic
nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage
caused by covalent modifications that typically distort and destabilize DNA
through the production of lesions derived from bulky chemical carcinogens, such
as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a
recent study challenges this lesion-recognition paradigm. The work of Vaisman et
al. (2013) [34] reveals that even a single ribonucleotide embedded in a
deoxyribonucleotide duplex is recognized by the bacterial NER machinery in
vitro. In their report, the authors show that spontaneous mutagenesis promoted
by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking
rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain
lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC
proteins in in vitro assays they show that despite causing little distortion, a
single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised
by the NER complex. We present the hypothesis to explain the recognition and/or
verification of this small lesion, that the critical 2'-OH of the ribonucleotide
- with its unique electrostatic and hydrogen bonding properties - may act as a
signal through interactions with amino acid residues of the prokaryotic NER
complex that are not possible with DNA. Such a mechanism might also be relevant
if it were demonstrated that the eukaryotic NER machinery likewise incises an
embedded ribonucleotide in DNA. In addition to its prominence in producing genetic diversity in bacterial
species, homologous recombination (HR) plays a key role in DNA repair and damage
tolerance. The frequency of HR depends on several factors, including the
efficiency of DNA repair systems as HR is involved in recovery of replication
forks perturbed by DNA damage. Nucleotide excision repair (NER) is one of the
major DNA repair pathways involved in repair of a broad range of DNA lesions
generally induced by exogenous chemicals or UV-irradiation and its functions in
the cells not exposed to DNA-damaging agents have attracted less attention. In
this study we have developed an assay that enables to investigate HR between
chromosomal loci of the soil bacterium Pseudomonas putida both in growing and
stationary-phase cells. The present assay detects HR events between two
non-functional alleles of phenol degrading genes that produce a functional
allele and allow the growth of bacteria on phenol as a sole carbon source. Our
results indicate that HR between chromosomal loci takes place mainly in the
growing cells and the frequency of HR is reduced during the following starvation
in NER-proficient P. putida but not in the case when bacteria lack UvrA or UvrB
enzymes. The absence of UvrA or UvrB resulted in a hyper-recombination phenotype
in P. putida, the cells were filamented and their growth was impaired even in
the absence of exogenous DNA damage. However, NER-deficient derivatives that
overcame growth defects emerged rapidly. Such adaptation resulted in the decline
of the frequency of HR. Although HR in actively replicating P. putida was still
elevated in the adapted variants of the UvrA- and UvrB-deficient strains, the
dynamics of emergence of the recombits in these strains turned similar to
NER-proficient bacteria. Additionally, we observed that HR was enhanced in the
absence of the transcription repair coupling factor Mfd in growing cells but not
during starvation. The frequency of HR was not affected by the UvrA homologue
UvrA2 neither in NER-proficient bacteria nor in the absence of UvrA, suggesting
a minor role of UvrA2 in NER. Thus, we conclude that NER functions are important
also without exogenously induced DNA damage in P. putida and both
transcription-coupled and global genome NER act to suppress HR in growing cells,
whereas UvrA and UvrB are involved in the maintece of the genome integrity
also in stationary-phase cells. |
Is JTV519 (K201) a potential drug for the prevention of arrhythmias? | Yes, JTV519 has antiarrhythmic properties. | 1. We investigated the effects of JTV-519
(4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,
5-tetrahydro-1,4-benzothiazepine monohydrochloride), a novel cardioprotective
drug, on the repolarizing K(+) currents in guinea-pig atrial cells by use of
patch-clamp techniques. We also evaluated the effects of JTV-519 on experimental
atrial fibrillation (AF) in isolated guinea-pig hearts. 2. In atrial cells
stimulated at 0.2 Hz, JTV-519 in concentrations of 0.3 and 1 microM slightly
prolonged the action potential duration (APD). The drug also reversed the action
potential shortening induced by the muscarinic agonist carbachol in a
concentration-dependent manner. 3. The muscarinic acetylcholine
receptor-operated K(+) current (I(K.ACh)) was activated by the extracellular
application of carbachol (1 microM), adenosine (10 microM) or by the
intracellular loading of GTP gamma S (100 microM). JTV-519 inhibited the
carbachol-, adenosine- and GTP gamma S-induced I(K.ACh) with the IC(50) values
of 0.12, 2.29 and 2.42 microM, respectively, suggesting that the drug may
inhibit I(K.ACh) mainly by blocking the muscarinic receptors. 4. JTV-519 (1
microM) inhibited the delayed rectifier K(+) current (I(K)).
Electrophysiological analyses indicated that the drug preferentially inhibits
I(Kr) (rapidly activating component) but not I(Ks) (slowly activating
component). 5. In isolated hearts, perfusion of carbachol (1 microM) shortened
monophasic action potential (MAP) and effective refractory period (ERP), and
lowered atrial fibrillation threshold (AFT). Addition of JTV-519 (1 microM)
inhibited the induction of AF by prolonging MAP and ERP. 6. We conclude that
JTV-519 can exert antiarrhythmic effects against AF by inhibiting repolarizing
K(+) currents. The drug may be useful for the treatment of AF in patients with
ischaemic heart disease. Effect of JTV-519 on AF.
INTRODUCTION: A new cardioprotective drug, JTV-519, blocks Na+ current and
inwardly rectifying K+ current and inhibits Ca2+ current. However, its role in
atrial electrophysiology is unknown. We investigated the antiarrhythmic effects
of JTV-519 on atrial fibrillation/flutter in the canine sterile pericarditis
model.
METHODS AND RESULTS: In nine dogs with sterile pericarditis, 38 episodes of
sustained (>30 sec) atrial fibrillation (8 dogs) and 24 episodes of sustained
atrial flutter (7 dogs) were induced by rapid atrial pacing. When atrial
fibrillation or atrial flutter was sustained >15 minutes, it was cardioverted
and reinduced. The inducibility of atrial fibrillation/flutter, the atrial
effective refractory period, and the intra-atrial conduction time were compared
before and after the continuous infusion of JTV-519 (0.03 mg/kg/min). JTV-519
significantly decreased the mean number of sustained atrial fibrillation
episodes (from 4.2 +/- 2.9 to 0 +/- 0, P < 0.01). In contrast, atrial flutter
was still inducible in 4 dogs after JTV-519 (from 2.7 +/- 2.5 to 1.6 +/- 2.1, P
= NS). JTV-519 significantly prolonged effective refractory period (from 123 +/-
18 to 143 +/- 14 msec, from 127 +/- 18 to 151 +/- 12 msec, and from 132 +/- 13
to 159 +/- 9 msec at basic cycle lengths of 200, 300, and 400 msec,
respectively, P < 0.01), but it did not affect the intra-atrial conduction time
(from 47 +/- 11 msec to 48 +/- 11 msec, P = NS).
CONCLUSION: JTV-519 had significant protective effects on atrial fibrillation in
the canine sterile pericarditis model, mainly by increasing effective refractory
period, suggesting that it may have potential as a novel antiarrhythmic agent
for atrial fibrillation. Ventricular arrhythmias can cause sudden cardiac death (SCD) in patients with
normal hearts and in those with underlying disease such as heart failure. In
animals with heart failure and in patients with inherited forms of
exercise-induced SCD, depletion of the channel-stabilizing protein calstabin2
(FKBP12.6) from the ryanodine receptor-calcium release channel (RyR2) complex
causes an intracellular Ca2+ leak that can trigger fatal cardiac arrhythmias. A
derivative of 1,4-benzothiazepine (JTV519) increased the affinity of calstabin2
for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak
that triggers arrhythmias. Thus, enhancing the binding of calstabin2 to RyR2 may
be a therapeutic strategy for common ventricular arrhythmias. Calstablin2 stabilises the ryanodine receptor (RyR2), preventing aberrant
activation of the channels during the resting phase of the cardiac muscle. Loss
of this stabilisation may be associated with cardiac arrhythmias, the sudden
death occasionally observed in people with structurally normal hearts, as well
as the atrial fibrillation in heart failure. Calstabin2-deficient mice have
structurally normal hearts but exhibit exercise-induced cardiac ventricular
arrhythmias that cause sudden death. In arrhythmias, the calstabin2 stabiliser
JTV519 did not prevent arrhythmias in calstabin2-/- mice, but reduced the
arrhythmias in calstabin2+/- mice, illustrating the antiarrhythmic potential of
stabilising calstablin2. Familial polymorphic ventricular tachycardia in humans
has been linked to missense mutants in the hRyR2 gene. In HEK293 cells, these
RyR2 mutants showed less binding of 35S-calstabin2 than the wild type,
indicating a reduced binding affinity. In human atrial fibrillation and heart
failure, where there is excessive disassociation of calstabin2 from the RyR2
receptor in vitro, JTV519 is able to reverse this. In conclusion, calstabin2 is
an important new target in sudden cardiac death associated with structurally
normal hearts, and in the treatment of atrial fibrillation and heart failure. Catecholaminergic polymorphic ventricular tachycardia is a form of
exercise-induced sudden cardiac death that has been linked to mutations in the
cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the
sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic
ventricular tachycardia-linked RyR2 mutations significantly decrease the binding
affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state
of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak
triggers ventricular tachycardia (VT) and sudden cardiac death. In
calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak,
monophasic action potential alters, and bidirectional VT. Calstabin-deficient
cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents
in diastole consistent with delayed after-depolarizations. The
1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2
for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential
alters and triggered arrhythmias. Our data suggest that calstabin-2
deficiency is as a critical mediator of triggers that initiate cardiac
arrhythmias. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
disease characterized by life threatening arrhythmias and mutations in the gene
encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2
mutations induce abnormal calcium transients in the absence of adrenergic
stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3)
K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied
ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring
the R4496C mutation (RyR2(R4496C+/-)). Pacing protocols did not elicit delayed
afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%)
RyR2(R4496C+/-) myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L)
induced small DADs (45%) and no triggered activity in WT myocytes, whereas it
elicited DADs in 87% and triggered activity in 60% of RyR2(R4496C+/-) myocytes
(P=0.001). DADs and triggered activity were abolished by ryanodine (10
micromol/L) but not by K201 (1 micromol/L or 10 micromol/L). In vivo
administration of K201 failed to prevent induction of polymorphic ventricular
tachycardia (VT) in RyR2(R4496C+/-) mice. Measurement of the FKBP12.6/RyR2 ratio
in the heavy sarcoplasmic reticulum membrane showed normal RyR2-FKBP12.6
interaction both in WT and RyR2(R4496C+/-) either before and after treatment
with caffeine and epinephrine. We suggest that (1) triggered activity is the
likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in
RyR2(R4496C+/-) myocytes and ventricular arrhythmias in RyR2(R4496C+/-) mice;
and (3) RyR2-FKBP12.6 interaction in RyR2(R4496C+/-) is identical to that of WT
both before and after epinephrine and caffeine, thus suggesting that it is
unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex. OBJECTIVES: The drug K201 (JTV-519) increases inotropy and suppresses
arrhythmias in failing hearts, but the effects of K201 on normal hearts is
unknown.
METHODS: The effect of K201 on excitation-contraction (E-C) coupling in normal
myocardium was studied by using voltage-clamp and intracellular Ca(2+)
measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed
using permeabilised cardiomyocytes.
RESULTS: Acute application of <1 micromol/L K201 had no significant effect on
E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to
83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L
K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with
accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content
(74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by
increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of
spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and
Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by
confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of
SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201
completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to
approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and
frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that
K201 inhibited both SR Ca(2+) uptake and release.
CONCLUSIONS: K201 modifies E-C coupling in normal cardiomyocytes. A dual
inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress
spontaneous diastolic Ca(2+) release during Ca(2+) overload without
significantly affecting Ca(2+) transient amplitude. BACKGROUND AND PURPOSE: Pulmonary veins are the most important focus for the
generation of atrial fibrillation. Abnormal calcium homeostasis with ryanodine
receptor dysfunction may underlie the arrhythmogenic activity in pulmonary
veins. The preferential ryanodine receptor stabilizer (K201) possesses
antiarrhythmic effects through calcium regulation. The purpose of this study was
to investigate the effects of K201 on the arrhythmogenic activity and calcium
regulation of pulmonary vein cardiomyocytes.
EXPERIMENTAL APPROACH: The ionic currents and intracellular calcium were studied
in isolated single cardiomyocytes from rabbit pulmonary vein before and after
the administration of K201, by the whole-cell patch clamp and indo-1
fluorimetric ratio techniques.
KEY RESULTS: K201 (0.1, 0.3, 1 microM) reduced the firing rates in pulmonary
vein cardiomyocytes, decreased the amplitudes of the delayed
afterdepolarizations and prolonged the action potential duration. K201 decreased
the L-type calcium currents, Na(+)/Ca(2+) exchanger currents, transient inward
currents and calcium transients. K201 (1 microM, but not 0.1 microM or 0.3
microM) also reduced the sarcoplasmic reticulum calcium content. Moreover, both
the pretreatment and administration of K201 (0.3 microM) decreased the
isoprenaline (10 nM)-induced arrhythmogenesis in pulmonary veins.
CONCLUSIONS AND IMPLICATIONS: K201 reduced the arrhythmogenic activity of
pulmonary vein cardiomyocytes and attenuated the arrhythmogenicity induced by
isoprenaline. These findings may reveal the anti-arrhythmic potential of K201. AIMS: Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2,
underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an
inherited life-threatening arrhythmia. CPVT is triggered by spontaneous
RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR
Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR
Ca(2+) content--in the absence of protein kinase A activation--affects RyR2
function and arrhythmogenesis in CPVT remains elusive.
METHODS AND RESULTS: Isolated murine ventricular myocytes harbouring a human
RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the
absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100
micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+)
load. Changes in membrane potential and intracellular [Ca(2+)] were monitored
with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At
baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+)
release, and SR Ca(2+) load were comparable in wild-type (WT) and
RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic
[Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+)
content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes.
Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves,
delayed afterdepolarizations and spontaneous APs, in both groups. However, the
ouabain-induced increase in the frequency of arrhythmogenic events was
dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519
greatly reduced the frequency of ouabain-induced arrhythmogenic events.
CONCLUSION: The elevation of SR Ca(2+) load--in the absence of beta-adrenergic
stimulation--is sufficient to increase the propensity for triggered arrhythmias
in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively
reduces these triggered arrhythmias. We propose that stabilizing ryanodine receptor type 2 (RyR2) may be a novel
strategy for the treatment of atrial fibrillation (AF). Sarcoplasmic reticulum
(SR) dysfunction caused by hyperphosphorylation of RyR2 and/or partial depletion
of the stabilizing subunit calstabin2 has been indicated in AF. RyR2
stabilization may prevent SR dysfunction and thereby protect the heart from
damage. Hypertrophy, hyperthyroidism and heart failure, three common
pathological conditions associated with AF, result in increased SR Ca(2+) leak
via RyR2. RyR2 stabilization may prevent SR Ca(2+) leak and thereby prevent AF.
Triggered activity appears to underlie atrial ectopic foci, which cause AF.
Diastolic Ca(2+) leak from SR via RyR2 may initiate triggered activity.
Therefore, modulating RyR2 opening probability would be predicted to protect
against triggered activity. Atrial oxidative stress plays a contributing role in
the pathogenesis of AF. RyR2 is a target of reactive oxygen species, and chronic
RyR2 oxidation increases RyR2 opening probability in a more sustained, less
reversible manner. Strategies that attenuate oxidative stress and protect
against AF may also contribute to RyR2 stabilization. Finally, KN-3 and JTV519,
two compounds that stabilize RyR2 in the closed state, prevent the induction of
triggered activity and suppress the inducibility of sustained AF. Thus, it is
reasonable to speculate that experimental approaches designed to improve RyR2
stabilization will drive a novel conceptual revolution in AF drug development
and lead to new clinical investigations. The ryanodine receptor (RyR) calcium release channel is an essential
intracellular ion channel that is central to Ca(2+) signaling and contraction in
the heart and skeletal muscle. The rapid release of Ca(2+) from the internal
sarcoplasmic reticulum Ca(2+) stores through the RyR during
excitation-contraction coupling is facilitated by the unique arrangement of the
surface and sarcoplasmic reticulum membrane systems. Debilitating and sometimes
fatal skeletal and cardiomyopathies result from changes in RyR activity that
disrupt normal Ca(2+) signaling. Such changes can be caused by point mutations
in many different regions of the RyR protein or acquired as a result of stress
associated with exercise, heart failure, age or drugs. In general, both
inherited and acquired changes include an increase in RyR channel activity.
Because of its central function, the RyR is a potential therapeutic target for
the inherited disorders and many of the acquired disorders. The RyR is currently
used as a therapeutic target in maligt hyperthermia where dantrolene is
effective and to relieve ventricular arrhythmia, with the use of JTV519 and
flecainide. These drugs show that the RyR is a valid therapeutic target, but
have side effects that prevent their chronic use. Thus there is an urgent need
for the development of skeletal and cardiac specific drugs to treat these
diverse muscle disorders. In this review, we discuss the mutations that cause
skeletal myopathies and cardiac arrhythmias and how these mutations pinpoint
residues within the RyR protein that are functionally significant and might be
developed as targets for therapeutic drugs. Metabolic syndrome is characterized by a combination of obesity, hypertension,
insulin resistance, dyslipidemia, and impaired glucose tolerance. This
multifaceted syndrome is often accompanied by a hyperdynamic circulatory state
characterized by increased blood pressure, total blood volume, cardiac output,
and metabolic tissue demand. Experimental, epidemiological, and clinical studies
have demonstrated that patients with metabolic syndrome have significantly
elevated cardiovascular morbidity and mortality rates. One of the main and
frequent complications seen in metabolic syndrome is cardiovascular disease. The
primary endpoints of cardiometabolic risk are coronary and peripheral arterial
disease, myocardial infarction, congestive heart failure, arrhythmia, and
stroke. Alterations in expression and/or functioning of several key proteins
involved in regulating and maintaining ionic homeostasis can cause cardiac
disturbances. One such group of proteins is known as ryanodine receptors
(intracellular calcium release channels), which are the major channels through
which Ca(2+) ions leave the sarcoplasmic reticulum, leading to cardiac muscle
contraction. The economic cost of metabolic syndrome and its associated
complications has a significant effect on health care budgets. Improvements in
body weight, blood lipid profile, and hyperglycemia can reduce cardiometabolic
risk. However, constant hyperadrenergic stimulation still contributes to the
burden of disease. Normalization of the hyperdynamic circulatory state with
conventional therapies is the most reasonable therapeutic strategy to date.
JTV519 (K201) is a newly developed 1,4-benzothiazepine drug with antiarrhythmic
and cardioprotective properties. It appears to be very effective in not only
preventing but also in reversing the characteristic myocardial changes and
preventing lethal arrhythmias. It is also a unique candidate to improve
diastolic heart failure in metabolic syndrome. AIM: Ventricular arrhythmia (VA) is a risk for sudden death. Polymorphic
ventricular tachycardia (VT) degenerating to ventricular fibrillation occurs
subsequent to the prolongation of the QT interval following administration of
catecholamines under Ca(2+) loading. Fatal VA also occurs in ischemia and
ischemic-reperfusion. We compared the suppressive effect of K201 (JTV519), a
multiple-channel blocker and cardiac ryanodine receptor-calcium release channel
(RyR2) stabilizer, with that of diltiazem, a Ca(2+ )channel blocker, in 2
studies of isoproterenol-induced (n = 30) and ischemic-reperfusion-induced VAs
(n = 38) in rats.
METHODS: Adult male Wistar rats were administered 12 mg/kg/min calcium chloride
(CaCl(2)) for 20 minutes and then 6 μg/kg/min isoproterenol was infused with
CaCl(2) for a further 20 minutes. In other rats, the left coronary artery was
ligated for 5 minutes followed by reperfusion for 20 minutes. K201 or diltiazem
(both 1 mg/kg) was administered before infusion of the isoproterenol or
induction of ischemia.
RESULTS: After administration of isoproterenol under Ca(2+) loading, fatal VA
frequently occurred in the vehicle (9 of 10 animals, 90%) and diltiazem (8 of
10, 80%) groups, and K201 significantly suppressed the incidences of arrhythmia
and mortality (2 of 10, 20%). In the reperfusion study, the incidence and the
time until occurrence of reperfusion-induced VA and mortality were significantly
suppressed in the K201 (2 of 15 animals, 13%) and diltiazem (1 of 9 animals,
11%) groups compared to the vehicle group (8 of 14 animals, 57%).
SIGNIFICANCE: Induction of VA in an experimental model was achieved with a low
dose of isoproterenol under Ca(2+) loading. K201 markedly suppressed both the
isoproterenol-induced and the reperfusion-induced VAs, whereas diltiazem did not
suppress the isoproterenol-induced VA. The results suggest that both VAs are
related to early after depolarization (EAD) and indicate that K201 has the
potential to suppress EAD by stabilizing RyR2 to mediate Ca(2+) release from the
sarcoplasmic reticulum and acting as a multiple-channel blocker. |
What is the mechanism of microRNA deregulation in carcinogenesis? | MicroRNAs (miRNAs) are endogenous non-protein coding single-stranded RNAs (19–25 nucleotides in length) generated from cleavage of larger non-coding RNAs by the ribonuclease III enzyme Dicer. They become part of the RNA-induced silencing complex and negatively regulate gene expression by binding to homologous 3'-UTR region of target protein-coding mRNAs as an imperfect match, causing translational repression or degradation. Approximately one-third of the protein-coding genes are susceptible to miRNA regulation. Accumulating evidence indicates that deregulated miRNA expression is associated with the onset and progression of a number of human cancers. Therefore, cancer-associated miRNAs (CA-miRNAs) could regulate target genes by acting either as "oncogenes" or "tumor suppressor miRNA (TS-miRNAs)". In line with this, numerous cancers (e.g. breast, lung, oesophageal, prostate, pancreatic, gastric and colon cancer) have been classified based on their unique miRNA expression profile. | MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles
by repressing translation or cleaving RNA transcripts. Although the number of
verified human miRNA is still expanding, only few have been functionally
described. However, emerging evidences suggest the potential involvement of
altered regulation of miRNA in pathogenesis of cancers and these genes are
thought to function as both tumours suppressor and oncogenes. In our study, we
examined by Real-Time PCR the expression of 156 mature miRNA in colorectal
cancer. The analysis by several bioinformatics algorithms of colorectal tumours
and adjacent non-neoplastic tissues from patients and colorectal cancer cell
lines allowed identifying a group of 13 miRNA whose expression is significantly
altered in this tumor. The most significantly deregulated miRNA being miR-31,
miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression
level of miR-31 was correlated with the stage of CRC tumor. Our results suggest
that miRNA expression profile could have relevance to the biological and
clinical behavior of colorectal neoplasia. Breast cancer is the most common maligcy in women continuing to rise
worldwide. Breast cancer emerges through a multi-step process, encompassing
progressive changes from a normal cell to hyperplasia (with and without atypia),
carcinoma in situ, invasive carcinoma, and metastasis. In the current study, we
analyzed the morphological changes and alterations of DNA methylation, histone
methylation and microRNA expression during estradiol-17beta (E(2))-induced
mammary carcinogenesis in female August Copenhagen Irish (ACI) rats.
E(2)-induced breast carcinogenesis in ACI rats provides a physiologically
relevant and genetically defined animal model for studying human sporadic breast
cancer. The pattern of morphological changes in mammary glands during
E(2)-induced carcinogenesis was characterized by transition from normal
appearing alveolar and ductular hyperplasia to focal hyperplastic areas of
atypical glands and ducts accompanied by a rapid and sustained loss of global
DNA methylation, LINE-1 hypomethylation, loss of histone H3 lysine 9 and histone
H4 lysine 20 trimethylation, and altered microRNAs expression. More importantly,
these alterations in the mammary tissue occurred after six weeks of
E(2)-treatment, whereas the atypical hyperplasia, which represents a putative
precursor lesion to mammary carcinoma in this model, was detected only after
twelve weeks of exposure, demonstrating clearly that these events are directly
associated with the effects of E(2) and are not a consequence of the preexisting
preneoplastic lesions. The results of this study show that deregulation of
cellular epigenetic processes plays a crucial role in the mechanism of
E(2)-induced mammary carcinogenesis in ACI rats, especially in the tumor
initiation process. MicroRNAs are a recently discovered class of small noncoding functional RNAs.
These molecules mediate post-transcriptional regulation of gene expression in a
sequence specific manner. MicroRNAs are now known to be key players in a variety
of biological processes and have been shown to be deregulated in a number of
cancers. The discovery of viral encoded microRNAs, especially from a family of
oncogenic viruses, has attracted immense attention towards the possibility of
microRNAs as critical modulators of viral oncogenesis. The host-virus crosstalk
mediated by microRNAs, messenger RNAs and proteins, is complex and involves the
different cellular regulatory layers. In this commentary, we describe models of
microRNA mediated viral oncogenesis. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression
post-transcriptionally. After the discovery of the first miRNA in the roundworm
Caenorhabditis elegans, these short regulatory RNAs have been found to be an
abundant class of RNAs in plants, animals, and DNA viruses. About 3% of human
genes encode for miRNAs, and up to 30% of human protein coding genes may be
regulated by miRNAs. MicroRNAs play a key role in diverse biological processes,
including development, cell proliferation, differentiation, and apoptosis.
Accordingly, altered miRNA expression is likely to contribute to human disease,
including cancer. This review will summarize the emerging knowledge of the
connections between human miRNA biology and different aspects of carcinogenesis.
Various techniques available to investigate miRNAs will also be discussed. BACKGROUND: The role of microRNAs (miRNAs) in multiple myeloma (MM) has yet to
be fully elucidated. To identify miRNAs that are potentially deregulated in MM,
we investigated those mapping within transcription units, based on evidence that
intronic miRNAs are frequently coexpressed with their host genes. To this end,
we monitored host transcript expression values in a panel of 20 human MM cell
lines (HMCLs) and focused on transcripts whose expression varied significantly
across the dataset.
METHODS: miRNA expression was quantified by Quantitative Real-Time PCR. Gene
expression and genome profiling data were generated on Affymetrix
oligonucleotide microarrays. Significant Analysis of Microarrays algorithm was
used to investigate differentially expressed transcripts. Conventional
statistics were used to test correlations for significance. Public libraries
were queried to predict putative miRNA targets.
RESULTS: We identified transcripts specific to six miRNA host genes (CCPG1,
GULP1, EVL, TACSTD1, MEST, and TNIK) whose average changes in expression varied
at least 2-fold from the mean of the examined dataset. We evaluated the
expression levels of the corresponding intronic miRNAs and identified a
significant correlation between the expression levels of MEST, EVL, and GULP1
and those of the corresponding miRNAs miR-335, miR-342-3p, and miR-561,
respectively. Genome-wide profiling of the 20 HMCLs indicated that the increased
expression of the three host genes and their corresponding intronic miRNAs was
not correlated with local copy number variations. Notably, miRNAs and their host
genes were overexpressed in a fraction of primary tumors with respect to normal
plasma cells; however, this finding was not correlated with known molecular
myeloma groups. The predicted putative miRNA targets and the transcriptional
profiles associated with the primary tumors suggest that MEST/miR-335 and
EVL/miR-342-3p may play a role in plasma cell homing and/or interactions with
the bone marrow microenvironment.
CONCLUSION: Our data support the idea that intronic miRNAs and their host genes
are regulated dependently, and may contribute to the understanding of their
biological roles in cancer. To our knowledge, this is the first evidence of
deregulated miRNA expression in MM, providing insights that may lead to the
identification of new biomarkers and altered molecular pathways of the disease. 'miR2Disease', a manually curated database, aims at providing a comprehensive
resource of microRNA deregulation in various human diseases. The current version
of miR2Disease documents 1939 curated relationships between 299 human microRNAs
and 94 human diseases by reviewing more than 600 published papers. Around
one-seventh of the microRNA-disease relationships represent the pathogenic roles
of deregulated microRNA in human disease. Each entry in the miR2Disease contains
detailed information on a microRNA-disease relationship, including a microRNA
ID, the disease name, a brief description of the microRNA-disease relationship,
an expression pattern of the microRNA, the detection method for microRNA
expression, experimentally verified target gene(s) of the microRNA and a
literature reference. miR2Disease provides a user-friendly interface for a
convenient retrieval of each entry by microRNA ID, disease name, or target gene.
In addition, miR2Disease offers a submission page that allows researchers to
submit established microRNA-disease relationships that are not documented. Once
approved by the submission review committee, the submitted records will be
included in the database. miR2Disease is freely available at
http://www.miR2Disease.org. BACKGROUND: MicroRNAs are a family of 19- to 25-nucleotides noncoding small RNAs
that primarily function as gene regulators. Aberrant microRNA expression has
been described for several human maligcies, and this new class of small
regulatory RNAs has both oncogenic and tumor suppressor functions. Despite this
knowledge, there is little information regarding microRNAs in plasma especially
because microRNAs in plasma, if exist, were thought to be digested by RNase.
Recent studies, however, have revealed that microRNAs exist and escape digestion
in plasma.
METHODOLOGY/PRINCIPAL FINDINGS: We performed microRNA microaray to obtain
insight into microRNA deregulation in the plasma of a leukemia patient. We have
revealed that microRNA-638 (miR-638) is stably present in human plasmas, and
microRNA-92a (miR-92a) dramatically decreased in the plasmas of acute leukemia
patients. Especially, the ratio of miR-92a/miR-638 in plasma was very useful for
distinguishing leukemia patients from healthy body.
CONCLUSIONS/SIGNIFICANCE: The ratio of miR-92a/miR-638 in plasma has strong
potential for clinical application as a novel biomarker for detection of
leukemia. INTRODUCTION: MicroRNAs (miRNAs) are a class of small noncoding RNAs that
control gene expression by targeting mRNAs and triggering either translation
repression or RNA degradation. Their aberrant expression may be involved in
human diseases, including cancer. To test the hypothesis that there is a
specific miRNA expression signature which characterizes male breast cancers, we
performed miRNA microarray analysis in a series of male breast cancers and
compared them with cases of male gynecomastia and female breast cancers.
METHODS: Paraffin blocks were obtained at the Department of Pathology of Thomas
Jefferson University from 28 male patients including 23 breast cancers and five
cases of male gynecomastia, and from 10 female ductal breast carcinomas. The RNA
harvested was hybridized to miRNA microarrays (~1,100 miRNA probes, including
326 human and 249 mouse miRNA genes, spotted in duplicate). To further support
the microarray data, an immunohistochemical analysis for two specific miRNA gene
targets (HOXD10 and VEGF) was performed in a small series of male breast
carcinoma and gynecomastia samples.
RESULTS: We identified a male breast cancer miRNA signature composed of a large
portion of underexpressed miRNAs. In particular, 17 miRNAs with increased
expression and 26 miRNAs with decreased expression were identified in male
breast cancer compared with gynecomastia. Among these miRNAs, some had
well-characterized cancer development association and some showed a deregulation
in cancer specimens similar to the one previously observed in the published
signatures of female breast cancer. Comparing male with female breast cancer
miRNA expression signatures, 17 significantly deregulated miRNAs were observed
(four overexpressed and 13 underexpressed in male breast cancers). The HOXD10
and VEGF gene immunohistochemical expression significantly follows the
corresponding miRNA deregulation.
CONCLUSIONS: Our results suggest that specific miRNAs may be directly involved
in male breast cancer development and that they may represent a novel diagnostic
tool in the characterization of specific cancer gene targets. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene
expression by targeting messenger RNA (mRNA). Recently, it has been demonstrated
that miRNA expression is altered in many human diseases including cancers,
suggesting that miRNA may play a potential role in the pathogenesis of different
diseases. It has also been reported that there is a unique expression pattern of
miRNAs in the disease state differing from the normal state. In this review, we
focus on the miRNA signatures in different human diseases including cancers.
Such signatures may be used as diagnostic and prognostic markers. MicroRNAs are small non-coding RNAs that regulate gene expression at the
transcriptional or posttranscriptional level. They are involved in cellular
development, differentiation, proliferation and apoptosis and play a significant
role in cancer. Examination of tumor-specific microRNA expression profiles has
revealed widespread deregulation of these molecules in diverse cancers. Several
studies have shown that microRNAs function either as tumor suppressor genes or
oncogenes, whose loss or overexpression respectively has diagnostic and
prognostic significance. It seems that microRNAs act as major regulators of gene
expression. In this review, we discuss microRNAs' role in cancer and how
microRNAs exert their functions through regulation of their gene targets.
Bioinformatic analysis of putative miRNA binding sites has indicated several
novel potential gene targets involved in apoptosis, angiogenesis and metastatic
mechanisms. Matching computational prediction analysis together with microarray
data seems the best method for microRNA gene target identification. MicroRNAs
together with transcription factors generate a complex combinatorial code
regulating gene expression. Thus, manipulation of microRNA-transcription factor
gene networks may be provides a novel approach for developing cancer therapies. MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small
non-coding RNAs that are endogenously expressed with crucial functions in
fundamental cellular processes such as cell cycle, apoptosis and
differentiation. Disturbance of miRNA expression and function leads to
deregulation of basic cellular processes leading to tumorigenesis. A growing
body of experimental evidence suggests that human tumors have deregulated
expression of microRNAs, which have been proposed as novel oncogenes or tumor
suppressors. Recent studies have shown that microRNA expression patterns serve
as phenotypic signatures of different cancers and could be used as diagnostic,
prognostic and therapeutic tools. A few studies have analyzed global microRNA
expression profiles or the functional role of microRNAs in prostate cancer. Here
we have reviewed the role of microRNAs in prostate carcinogenesis by summarizing
the findings from such studies. In addition, recent evidence indicates that
dietary factors play an important role in the process of carcinogenesis through
modulation of miRNA expression, though such studies are lacking in regards to
prostate cancer. It has been proposed that dietary modulation of miRNA
expression may contribute to the cancer-protective effects of dietary
components. In this review, we have summarized findings from studies on the
effect of dietary agents on miRNA expression and function. BACKGROUND: Heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type
(hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating
in alternative splicing by antagonising other splicing factors, notably ASF/SF2.
The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone
in human lung cancer and their potential to serve as molecular markers for early
diagnosis remain issues of intense investigation. The main objective of the
present study was to use paired tumour/non-tumour biopsies from patients with
non-small cell lung cancer (NSCLC) to investigate the expression profiles of
hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2.
METHODS: We combined western blotting of tissue homogenates with
immunohistochemical examination of fixed tissue sections and quantification of
mRNA expression levels in tumour versus adjacent normal-looking areas of the
lung in the same patient.
RESULTS: Our study, in addition to clear evidence of mostly uncoupled
deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated
protein with a high frequency of over-expression (76%), followed by A3 (52%) and
A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of
correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1,
suggesting that different mechanisms underlie their deregulation.
CONCLUSION: Our results provide strong evidence for the up-regulation of hnRNP
A/B in NSCLC, and they support the existence of distinct mechanisms responsible
for their deregulated expression. BACKGROUND: Recently, microRNAs in cancer development have attracted much
attention, but their roles in tumorigenesis are still largely unknown. In this
study, a functional role of miR-22 in hepatocellular carcinoma (HCC) development
has been identified.
METHODS: Quantitative real-time PCR was used to determine the level of miR-22
transcript in HCC clinical samples, and its correlation with disease-free
survival was determined using Kaplan-Meier method. Restoration of miR-22
expression was carried out in HCC cell lines to assess its influence on HCC cell
proliferation and tumourigenicity.
RESULTS: In the 160 paired HCC tissue samples, miR-22 expression was
downregulated in HCC, and low miR-22 expression in HCC was predictive of poor
survival in HCC patients. Functional studies indicated that ectopic expression
of miR-22 significantly inhibits HCC cell proliferation and tumourigenicity.
Furthermore, histone deacetylase 4 (HDAC4), known to have critical roles in
cancer development, was proved to be directly targeted and regulated by miR-22.
Furthermore, HDAC4 was upregulated in miR-22-downregulated HCC tissues,
suggesting that downregulation of miR-22 might participate in HCC carcinogenesis
and progression through potentiation of HDAC4 expression. In addition, cell
proliferation was also suppressed by knockdown of HDAC4 or treatment with HDAC
inhibitor trichostatin A in HCC cell lines.
CONCLUSION: miR-22, downregulated in HCC, has an anti-proliferative effect on
HCC cells both in vitro and in vivo. Furthermore, miR-22 may have considerable
potential in identification of the prognosis and application of cancer therapy
for HCC patients. MicroRNAs are small noncoding RNAs that function to control gene expression.
These small RNAs have been shown to contribute to the control of cell growth,
differentiation and apoptosis, important features related to cancer development
and progression. In fact, recent studies have shown the utility of microRNAs as
cancer-related biomarkers. This is due to the finding that microRNAs display
altered expression profiles in cancers versus normal tissue. In addition,
microRNAs have been associated with cancer progression. In this review, the
mechanisms to alter microRNA expression and their relation to cancer will be
addressed. Moreover, the potential application of microRNAs in clinical settings
will also be highlighted. Finally, the challenges regarding the translation of
research involving microRNAs to the clinical realm will be discussed. BACKGROUND: Dysregulated expression of microRNAs (miRNAs) has been previously
observed in human cancer tissues and shown promise in defining tumor status.
However, there is little information as to if or when expression changes of
miRNAs occur in normal tissues after carcinogen exposure.
RESULTS: To explore the possible time-course changes of miRNA expression induced
by a carcinogen, we treated mice with one dose of 120 mg/kg
N-ethyl-N-nitrosourea (ENU), a model genotoxic carcinogen, and vehicle control.
The miRNA expression profiles were assessed in the mouse livers in a time-course
design. miRNAs were isolated from the livers at days 1, 3, 7, 15, 30 and 120
after the treatment and their expression was determined using a miRNA PCR Array.
Principal component analysis of the miRNA expression profiles showed that miRNA
expression at post-treatment days (PTDs) 7 and 15 were different from those at
the other time points and the control. The number of differentially expressed
miRNAs (DEMs) changed over time (3, 5, 14, 32, 5 and 5 at PTDs 1, 3, 7, 15, 30
and 120, respectively). The magnitude of the expression change varied with time
with the highest changes at PTDs 7 or 15 for most of the DEMs. In silico
functional analysis of the DEMs at PTDs 7 and 15 indicated that the major
functions of these ENU-induced DEMs were associated with DNA damage, DNA repair,
apoptosis and other processes related to carcinogenesis.
CONCLUSION: Our results showed that many miRNAs changed their expression to
respond the exposure of the genotoxic carcinogen ENU and the number and
magnitude of the changes were highest at PTDs 7 to 15. Thus, one to two weeks
after the exposure is the best time for miRNA expression sampling. 16th international charles heidelberger symposium on cancer research coimbra,
portugal. 26-28 september 2010. Abstracts. BACKGROUND: Gastric cancer is the fourth most common cancer in the world and the
second most prevalent cause of cancer related death. The development of gastric
cancer is mainly associated with H. Pylori infection leading to a focus in
pathology studies on bacterial and environmental factors, and to a lesser extent
on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA
molecules involved in post-transcriptional gene regulation. They are found to
regulate genes involved in diverse biological functions and alterations in
microRNA expression have been linked to the pathogenesis of many maligcies.
The current study is focused on identifying microRNAs involved in gastric
carcinogenesis and to explore their mechanistic relevance by characterizing
their targets.
RESULTS: Invitrogen NCode miRNA microarrays identified miR-449 to be decreased
in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared
to tissues from wild type animals. Growth rate of gastric cell lines
over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell
cycle analysis of miR-449 over-expressing cells showed a significant increase in
the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent
phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays
identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase
assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We
also show that miR-449 over-expression activated p53 and its downstream target
p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR
analyses showed a loss of miR-449 expression in human clinical gastric tumours
compared to normal tissues.
CONCLUSIONS: In this study, we document a diminished expression of miR-449 in
Gastrin KO mice and further confirmed its loss in human gastric tumours. We
investigated the function of miR-449 by identifying its direct targets.
Furthermore we show that miR-449 induces senescence and apoptosis by activating
the p53 pathway. Alterations in microRNAs expression have been proposed to play role in
endometrial cancer pathogenesis. Dicer and Drosha are main regulators of
microRNA biogenesis and deregulation of their expression has been indicated as a
possible cause of microRNAs alterations observed in various cancers. The
objective of this study was to investigate Dicer and Drosha genes expression in
endometrial cancer and to analyze the impact of clinicopathological
characteristics on their expression. Fresh tissue samples were collected from 44
patients (26 endometroid endometrial carcinoma and 18 controls). Clinical and
pathological data were acquired from medical documentation. Dicer and Drosha
genes expressions were assessed by qRT-PCR using validated reference genes.
Dicer and Drosha expression levels were significantly lower in endometrial
cancer samples comparing to controls. Dicer was down-regulated by the factor of
1.54 (p=0.009) and Drosha gene mean expression value was 1.4 times lower in
endometrial cancer group versus control group (p=0.008). Down-regulation of
Dicer significantly correlated with decreased expression of Drosha (coefficient
value 0.75). Decreased expression of Drosha correlated with higher histological
grade and was influenced by BMI. Lower Dicer expression was found in nulli- and
uniparous females comparing to multiparous individuals (p=0.002). Neither the
FIGO stage nor the menstrual status had significant influence on the expression
of studied genes. This study revealed for the first time that expression
alterations of main regulators of microRNAs biogenesis are present in
endometrial cancer tissue and could be potentially responsible for altered
microRNAs profiles observed in this maligcy. Glioblastoma is the most common and most lethal brain tumour in humans.
Illustrating the functions being disturbed during carcinogenesis and how they
are deregulated is very important for us to understand its underlying mechanism.
Transcriptional aberrations may play a vital role in the etiology of
glioblastoma, which might be caused by both genomic alterations and other
regulation molecules. In this study, we investigated possible cooperative
deregulation of microRNAs (miRNAs) and transcription factors (TFs) in
glioblastoma, under the hypothesis that miRNAs and TFs might have a
combinational regulatory effect on glioblastoma genes. We searched
glioblastoma-specific regulatory networks by integrating glioblastoma related
miRNAs, TFs and genes, and identified 54 feed-forward loops (FFLs). Follow up
functional enrichment analysis of these FFLs uncovered some functions important
to carcinogenesis but also some unique functions specific to the FFLs we
identified. MicroRNAs (miRNAs) play an important role in carcinogenesis through the
regulation of their target genes. miRNA-related single nucleotide polymorphisms
(miR-SNPs) can affect miRNA biogenesis and target sites and can alter microRNA
expression and functions. We examined 11 miR-SNPs, including 5 in microRNA
genes, 3 in microRNA binding sites and 3 in microRNA-processing machinery
components, and evaluated time to recurrence (TTR) according to miR-SNP
genotypes in 175 surgically resected non-small-cell lung cancer (NSCLC)
patients. Significant differences in TTR were found according to KRT81 rs3660
(median TTR: 20.3 months for the CC genotype versus 86.8 months for the CG or GG
genotype; P = 0.003) and XPO5 rs11077 (median TTR: 24.7 months for the AA
genotype versus 73.1 months for the AC or CC genotypes; P = 0.029). Moreover,
when patients were divided according to stage, these differences were maintained
for stage I patients (P = 0.002 for KRT81 rs3660; P<0.001 for XPO5 rs11077).
When patients were divided into sub-groups according to histology, the effect of
the KRT81 rs3660 genotype on TTR was significant in patients with squamous cell
carcinoma (P = 0.004) but not in those with adenocarcinoma. In the multivariate
analyses, the KRT81 rs3660 CC genotype (OR = 1.8; P = 0.023) and the XPO5
rs11077 AA genotype (OR = 1.77; P = 0.026) emerged as independent variables
influencing TTR. Immunohistochemical analyses in 80 lung specimens showed that
95% of squamous cell carcinomas were positive for KRT81, compared to only 19% of
adenocarcinomas (P<0.0001). In conclusion, miR-SNPs are a novel class of SNPs
that can add useful prognostic information on the clinical outcome of resected
NSCLC patients and may be a potential key tool for selecting high-risk stage I
patients. Moreover, KRT81 has emerged as a promising immunohistochemical marker
for the identification of squamous cell lung carcinoma. Lung cancers account for a huge percentage of death in industrialized countries,
and hence there is an increasing call for the development of novel treatments.
These maligcies are caused by a combination of environmental factors,
principally cigarette smoking and genetic alterations. MicroRNAs (miRNAs) are a
recently discovered class of regulatory noncoding small RNAs with a significance
in numerous biological processes. Strong evidence links miRNA impaired
expression profiles and pathways to the etiology of several diseases, including
neoplasia. This paper focuses on the emerging role of miRNA function in lung
cancer development with particular highlighting on the use of miRNA profiles and
polymorphisms for the molecular and biological characterization of tumor
pulmonary growth and progression. Furthermore, we underline the potential
utility of lung cancer-associated miRNAs as clinical biomarkers with a
diagnostic, prognostic, and therapeutic significance and give emphasis to the
promising novel miRNA-based curative strategies. Currently, carcinogenesis is considered to be the result of mal-expression of
tumour suppressor genes and oncogenes, leading either way to uncontrollable and
disorganized cell mitosis. Recently a novel class of genes has drawn the
interest of the scientific community. These are microRNAs (miRNAs), a class of
noncoding RNAs, 20-23 nucleotides in length, that can up or downregulate gene
expression of downstream gene targets (including transcription factors,
oncogenes, and tumour suppressor genes) at the post-transcriptional level. Some
members of this new class of genes seem to have the potential to act
simultaneously either as oncogenes or as tumour suppressor genes depending on
the molecular microenvironment of the cell. We elaborate on this hypothesis by
giving examples of miRNAs (e.g. mir-9, miR-17-92) which seem to function by the
abovementioned mechanism. This could mean that the deterministic notion of
carcinogenesis as a result of merely tumour suppressor genes and oncogenes
deregulation could be revised to contain the fact that certain members of this
novel class of genes have the potential to play both roles simultaneously. MicroRNAs are key regulators of various fundamental biological processes and,
although representing only a small portion of the genome, they regulate a much
larger population of target genes. Mature microRNAs (miRNAs) are single-stranded
RNA molecules of 20-23 nucleotide (nt) length that control gene expression in
many cellular processes. These molecules typically reduce the stability of
mRNAs, including those of genes that mediate processes in tumorigenesis, such as
inflammation, cell cycle regulation, stress response, differentiation, apoptosis
and invasion. MicroRNA targeting is mostly achieved through specific
base-pairing interactions between the 5' end ('seed' region) of the miRNA and
sites within coding and untranslated regions (UTRs) of mRNAs; target sites in
the 3' UTR diminish mRNA stability. Since miRNAs frequently target hundreds of
mRNAs, miRNA regulatory pathways are complex. Calin and Croce were the first to
demonstrate a connection between microRNAs and increased risk of developing
cancer, and meanwhile the role of microRNAs in carcinogenesis has definitively
been evidenced. It needs to be considered that the complex mechanism of gene
regulation by microRNAs is profoundly influenced by variation in gene sequence
(polymorphisms) of the target sites. Thus, individual variability could cause
patients to present differential risks regarding several diseases. Aiming to
provide a critical overview of miRNA dysregulation in cancer, this article
reviews the growing number of studies that have shown the importance of these
small molecules and how these microRNAs can affect or be affected by genetic and
epigenetic mechanisms. BACKGROUND: Perturbation of DNA methylation is frequent in cancers and has
emerged as an important mechanism involved in tumorigenesis. To determine how
DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA
immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify
differentially DNA methylation sequences between primary glioma and normal brain
tissue samples.
METHODS: MeDIP-chip technology was used to investigate the whole-genome
differential methylation patterns in glioma and normal brain tissues.
Subsequently, the promoter methylation status of eight candidate genes was
validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system.
Then, the epigenetically regulated expression of these genes and the potential
mechanisms were examined by chromatin immunoprecipitation and quantitative
real-time PCR.
RESULTS: A total of 524 hypermethylated and 104 hypomethylated regions were
identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated
regions were mapped to the promoters of known genes related to a variety of
important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A,
GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in
primary glioma and cell lines. Aberrant promoter methylation and changed histone
modifications were associated with their reduced expression in glioma. In
addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in
the 22q11.2 in glioma and induction of miR-185 over-expression reduced global
DNA methylation and induced the expression of the promoter-hypermethylated genes
in glioma cells by directly targeting the DNA methyltransferases 1.
CONCLUSION: These comprehensive data may provide new insights into the
epigenetic pathogenesis of human gliomas. microRNAs (miRs) are endogenous small non-coding RNAs that are aberrantly
expressed in various carcinomas. miR-152 and miR-148a have not been
comprehensively investigated in ovarian cancer. Thus, the aim of this study was
to identify the role of miR-152 and miR-148a in epithelial ovarian cancer. Total
RNA was extracted from tissues of 78 patients with epithelial ovarian cancer, 17
normal ovarian epithelium tissues and two ovarian cancer cell lines. Using
quantitative real-time PCR (qRT-PCR) followed by the 2-ΔΔCT method for
calculating the results, we found that the expression levels of miR-152 were
significantly decreased in ovarian cancer tissues compared to normal ovarian
epithelium tissues (p<0.05). However, although the expression of miR-148a was
also decreased in 65% of patients, no statistically significant difference in
expression was found. A strong correlation was found between the expression of
miR-152 and miR-148a (p<0.001, Pearson's correlation). The relationship between
miR-152 or miR-148a expression levels in ovarian cancer and clinicopathological
features, response to therapy and short-term survival was analyzed and the
results showed that no correlation existed. In addition, we found that both
miR-152 and miR-148a were down-regulated in ovarian cancer cell lines. After
miR-152 or miR-148a mimics were transfected into ovarian cancer cell lines, the
MTT cell proliferation assay showed that cell proliferation was significantly
inhibited. Taken together, miR-152 and miR-148a may be involved in the
carcinogenesis of ovarian cancer through deregulation of cell proliferation.
They may be novel biomarkers for early detection or therapeutic targets of
ovarian cancer. MicroRNAs (miRNAs) are believed to have fundamental roles in tumorigenesis and
have great potential for the diagnosis and treatment of cancer. However, the
roles of miRNAs in hepatocellular carcinogenesis are still not fully elucidated.
We investigated the aberrantly expressed miRNAs involved in hepatoma by
comparison of miRNA expression profiles in cancerous hepatocytes with normal
primary human hepatocytes, and 37 dysregulated miRNAs were screened out by
twofold change with a significant difference (P<0.05). Clustering analysis based
on 13 miRNAs with changes over 15-folds showed that the miRNA expression
patterns between the cancerous and normal hepatocytes were clearly different.
Among the 13 miRNAs, we found that miR-375 was significantly downregulated in
hepatocellular carcinoma (HCC) tissues and cell lines. Overexpression of miR-375
in liver cancer cells decreased cell proliferation, clonogenicity,
migration/invasion and also induced G1 arrest and apoptosis. To unveil the
molecular mechanism of miR-375-mediated phenotype in hepatoma cells described
above, we examined the putative targets using bioinformatics tools and found
that astrocyte elevated gene-1 (AEG-1) was a potential target of miR-375. Then
we demonstrated that miR-375 bound directly to the 3'-untranslated region of
AEG-1 and inhibited the expression of AEG-1. TaqMan quantitative reverse
transcriptase-PCR and western blot analysis showed that miR-375 expression was
inversely correlated with AEG-1 expression in HCC tissues. Knockdown of AEG-1 by
RNAi in HCC cells, similar to miR-375 overexpression, suppressed tumor
properties. Ectopic expression of AEG-1, conversely, could partially reverse the
antitumor effects of miR-375. In a mouse model, therapeutic administration of
cholesterol-conjugated 2'-O-methyl-modified miR-375 mimics (Chol-miR-375) could
significantly suppress the growth of hepatoma xenografts in nude mice. In
conclusion, our findings indicate that miR-375 targets AEG-1 in HCC and
suppresses liver cancer cell growth in vitro and in vivo, and highlight the
therapeutic potential of miR-375 in HCC treatment. BACKGROUND: MicroRNA expression is severely disrupted in carcinogenesis, however
limited evidence is available validating results from cell-line models in human
clinical cancer specimens. MicroRNA-21 (mir-21) and microRNA-143 (mir-143) have
previously been identified as significantly deregulated in a range of cancers
including cervical cancer. Our goal was to investigate the expression patterns
of several well-studied microRNA species in cervical samples and compare the
results to cell line samples.
METHODOLOGY/PRINCIPAL FINDINGS: We measured the expression of mir-21 and mir-143
in 142 formalin-fixed, paraffin embedded (FFPE) cervical biopsy tissue blocks,
collected from Dantec Oncology Clinic, Dakar, Senegal. MicroRNA expression
analysis was performed using Taqman-based real-time PCR assays. Protein
immunohistochemical staining was also performed to investigate target protein
expression on 72 samples. We found that mir-21 expression increased with
worsening clinical diagnosis but that mir-143 was not correlated with histology.
These observations were in stark contrast to previous reports involving cervical
cancer cell lines in which mir-143 was consistently down-regulated but mir-21
largely unaffected. We also identified, for the first time, that cytoplasmic
expression of Programmed Cell Death Protein 4 PDCD4; a known target of mir-21)
was significantly lower in women with invasive cervical carcinoma (ICC) in
comparison to those with cervical intraepithelial neoplasia (2-3) or carcinoma
in situ (CIN2-3/CIS), although there was no significant correlation between
mir-21 and PDCD4 expression, despite previous studies identifying PDCD4
transcript as a known mir-21 target.
CONCLUSIONS: Whilst microRNA biomarkers have a number of promising features,
more studies on expression levels in histologically defined clinical specimens
are required to investigate clinical relevance of discovery-based studies.
Mir-21 may be of some utility in predictive screening, given that we observed a
significant correlation between mir-21 expression level and worsening
histological diagnosis of cervical cancer. Inflammation represents the body's natural response to tissue damage; however,
chronic inflammation may activate cell proliferation and induce deregulation of
cell death in affected tissues. Chronic inflammation is an important factor in
the development of hepatocellular carcinoma (HCC), although the precise
underlying mechanism remains unknown. Epigenetic events, which are considered
key mechanisms in the regulation of gene activity states, are also commonly
deregulated in HCC. Here, we review the evidence that chronic inflammation might
deregulate epigenetic processes, thus promoting oncogenic transformation, and we
propose a working hypothesis that epigenetic deregulation is an underlying
mechanism by which inflammation might promote HCC development. In this scenario,
different components of the inflammatory response might directly and indirectly
induce changes in epigenetic machineries ('epigenetic switch'), including those
involved in setting and propagating normal patterns of DNA methylation, histone
modifications and non-coding RNAs in hepatocytes. We discuss the possibility
that self-reinforcing cross-talk between inflammation and epigenetic mechanisms
might amplify inflammatory signals and maintain a chronic state of inflammation
culminating in cancer development. The potential role of inflammation-epigenome
interactions in the emergence and maintece of cancer stem cells is also
discussed. MicroRNAs (miRNAs) are short non-coding RNAs that play critical roles in
numerous cellular processes through post-transcriptional regulating functions.
The aberrant role of miRNAs has been reported in a number of hematopoietic
maligcies including multiple myeloma (MM). In this review we summarize the
current knowledge on roles of miRNAs in the pathogenesis of MM. Prostate cancer (PCA) still represents a leading cause of death. An increasing
number of studies have documented that microRNAs (miRNAs), a subgroup of
non-coding RNAs with gene regulatory functions, are differentially expressed in
PCA respect to the normal tissue counterpart, suggesting their involvement in
prostate carcinogenesis and dissemination. Interestingly, it has been shown that
miRNAs undergo the same regulatory mechanisms than any other protein coding
gene, including epigenetic regulation. In turn, miRNAs can also affect the
expression of oncogenes and tumor suppressor genes by targeting effectors of the
epigenetic machinery, therefore indirectly affecting the epigenetic controls on
these genes. Among the genes that undergo this complex regulation, there is the
androgen receptor (AR), a key therapeutic target for PCA. This review will focus
on the role of epigenetically regulated and epigenetically regulating miRNAs in
PCA and on the fine regulation of AR expression, as mediated by this
miRNA-epigenetics interaction. Recent advances in the field of RNA research have provided compelling evidence
implicating microRNA (miRNA) and long non-coding RNA molecules in many diverse
and substantial biological processes, including transcriptional and
post-transcriptional regulation of gene expression, genomic imprinting, and
modulation of protein activity. Thus, studies of non-coding RNA (ncRNA) may
contribute to the discovery of possible biomarkers in human cancers. Considering
that the response to chemotherapy can differ amongst individuals, researchers
have begun to isolate and identify the genes responsible. Identification of
targets of this ncRNA associated with cancer can suggest that networks of these
linked to oncogenes or tumor suppressors play pivotal roles in cancer
development. Moreover, these ncRNA are attractive drug targets since they may be
differentially expressed in maligt versus normal cells and regulate
expression of critical proteins in the cell. This review focuses on ncRNAs that
are differently expressed in maligt tissue, and discusses some of challenges
derived from their use as potential biomarkers of tumor properties. MicroRNAs (miRNAs) are short non-coding RNAs that are involved in several
important biological processes through regulation of genes
post-transcriptionally. Carcinogenesis is one of the key biological processes
where miRNAs play important role in the regulation of genes. The miRNAs elicit
their effects by binding to the 3' untranslated region (3'UTR) of their target
mRNAs, leading to the inhibition of translation or the degradation of the mRNA,
depending on the degree of complementary base pairing. To-date more than 1,000
miRNAs are postulated to exist, although the field is moving rapidly. Currently,
miRNAs are becoming the center of interest in a number of research areas,
particularly in oncology, as documented by exponential growth in publications in
the last decade. These studies have shown that miRNAs are deregulated in a wide
variety of human cancers. Thus, it is reasonable to ask the question whether
further understanding on the role of miRNAs could be useful for diagnosis,
prognosis and predicting therapeutic response for prostate cancer (PCa).
Therefore, in this review article, we will discuss the potential roles of
different miRNAs in PCa in order to provide up-to-date information, which is
expected to stimulate further research in the field for realizing the benefit of
miRNA-targeted therapeutic approach for the treatment of metastatic castrate
resistant prostate cancer (mCRPC) in the near future because there is no
curative treatment for mCRPC at the moment. B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal
translocations leading to the deregulation of proto-oncogenes. MicroRNAs can
also be affected by chromosomal alterations and thus contribute to
carcinogenesis. The microRNA, miR-125b-1, is overexpressed in B-ALL cases with
the t(11;14)(q24;q32) translocation; therefore, we sought to determine the role
of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine
and human leukemic B-cell lines to show that miR-125b expression enhances
proliferation by targeting B-cell regulator of immunoglobulin heavy-chain
transcription (Bright)/ARID3a, an activator of immunoglobulin heavy-chain
transcription. Accordingly, this target gene was downregulated in B-ALL patients
with the t(11;14)(q24;q32) translocation. Repression of Bright/ARID3a blocked
differentiation and conferred a survival advantage to Ba/F3 cells under
interleukin-3 starvation. In addition, overexpression of miR-125b protected
pre-BI and leukemic B-cell lines from apoptosis by blockade of caspase
activation by a mechanism that was independent of p53 and BAK1. In summary,
miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its
repression, thus leading to a blockage in differentiation, increased
proliferation and inhibition of apoptosis. Endometrial cancer is the seventh most common cancer in women worldwide.
Therefore elucidation of the pathogenesis and development of effective treatment
for endometrial cancer are important. However, several aspects of the mechanism
of carcinogenesis in the endometrium remain unclear. Associations with genetic
variation and mutations of cancer-related genes have been shown, but these do
not provide a complete explanation. Therefore, in recent years, epigenetic
mechanisms that do not involve changes in DNA sequences have been examined.
Studies aimed at detection of aberrant DNA hypermethylation in cancer cells
present in microscopic amounts in vivo and application of the results to cancer
diagnosis have also started. Breakdown of the DNA mismatch repair mechanism is
thought to play a large role in the development of endometrial cancer, with
changes in the expression of the hMLH1 gene being particularly important.
Silencing of genes such as APC and CHFR, Sprouty 2, RASSF1A, GPR54, CDH1, and
RSK4 by DNA hypermethylation, onset of Lynch syndrome due to hereditary
epimutation of hMLH1 and hMSH2 mismatch repair genes, and regulation of gene
expression by microRNAs may also underlie the carcinogenic mechanisms of
endometrial cancer. Further understanding of these issues may permit development
of new therapies. INTRODUCTION: Experimental and clinical evidence points to a critical role of
progesterone and the nuclear progesterone receptor (PR) in controlling mammary
gland tumorigenesis. However, the molecular mechanisms of progesterone action in
breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are
short ribonucleic acids which have also been found to play a pivotal role in
cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is
poorly explored. In this study we explored progestin modulation of miRNA
expression in mammary tumorigenesis.
METHODS: We performed a genome-wide study to explore progestin-mediated
regulation of miRNA expression in breast cancer. miR-16 expression was studied
by RT-qPCR in cancer cell lines with silenced PR, signal transducer and
activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins.
Breast cancer cells were transfected with the precursor of miR-16 and
proliferation assays, Western blots or in vivo experiments were performed.
Target genes of miR-16 were searched through a bioinformatical approach, and the
study was focused on cyclin E. Reporter gene assays were performed to confirm
that cyclin E 3'UTR is a direct target of miR-16.
RESULTS: We found that nine miRNAs were upregulated and seven were downregulated
by progestin in mammary tumor cells. miR-16, whose function as a tumor
suppressor in leukemia has already been shown, was identified as one of the
downregulated miRNAs in murine and human breast cancer cells. Progestin induced
a decrease in miR-16 levels via the classical PR and through a hierarchical
interplay between Stat3 and the oncogenic transcription factor c-Myc. A search
for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator
cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR
region. We found that, similar to the molecular mechanism underlying
progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the
induction of cyclin E expression by progestin. Moreover, overexpression of
miR-16 abrogated the ability of progestin to induce cyclin E upregulation,
revealing that cyclin E is a novel target of miR-16 in breast cancer.
Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in
vitro and in vivo, demonstrating for the first time, a role for miR-16 as a
tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand
heregulin (HRG) downregulated the expression of miR-16, which then participates
in the proliferative activity of HRG in breast tumor cells.
CONCLUSIONS: In this study, we reveal the first progestin-regulated miRNA
expression profile and identify a novel role for miR-16 as a tumor suppressor in
progestin- and growth factor-induced growth in breast cancer. INTRODUCTION: miRNAs are very important regulators in biological processes such
as development, cellular differentiation, and carcinogenesis. Given the
important role of miRNAs in tumorigenesis and development, it is worth
investigating whether some miRNAs play roles in the anticancer mechanism of
flavonoids. However, such a role has not yet been reported. We previously
selected the promising anticancer agent 3,6-dihydroxyflavone (3,6-DHF) in
pharmacodynamic experiments, which may serve as a leading compound for
developing more potent anticancer drugs or chemopreventive supplements. The
present study aims to investigate the chemopreventive activities of 3,6-DHF
against mammary carcinogenesis.
METHODS: The experimental model of breast carcinogenesis was developed by
intraperitoneal injection of 1-methyl-1-nitrosourea (MNU). The bioavailability
of 3,6-DHF in rats was detected by HPLC. The expression of microRNA-34a
(miR-34a) and microRNA-21 (miR-21) was evaluated by real-time quantitative
RT-PCR. Cell apoptosis was analyzed by flow cytometry or terminal
deoxynucleotidyl transferase dUTP nick end-labeling assay. The mitochondrial
membrane potential was assayed using
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide dye by
confocal laser scanning microscopy. The level of cytochrome C in cytosol was
evaluated by western blotting.
RESULTS: Our study showed that oral administration of 3,6-DHF effectively
suppressed MNU-induced breast carcinogenesis in rats, decreasing the cancer
incidence by 35.7%. The detection of bioavailability indicated that the
concentration of 3,6-DHF was 2.5 ± 0.4 μg/ml in plasma of rats within 2 hours
after administration, and was 21.7 ± 3.8 μg/ml in urine within 24 hours. Oral
administration of 3,6-DHF to BALB/c nude mice bearing breast cancer cell
xenografts also significantly suppressed tumor growth in vivo. Furthermore, our
study revealed that the global upregulation of miR-21 and downregulation of
miR-34a in breast carcinogenesis could be reversed by 3,6-DHF, which
significantly upregulated miR-34a expression and decreased miR-21 expression -
inducing apoptosis of breast cancer cells in vitro and in vivo. Overexpression
of miR-34a induced by plasmid transfection or inhibition of miR-21 by
oligonucleotides markedly promoted the pro-apoptotic effect of 3,6-DHF.
Inactivation of miR-34a or overproduction of miR-21 compromised the anticancer
effects of 3,6-DHF.
CONCLUSION: These findings indicate that 3,6-DHF is a potent natural
chemopreventive agent, and that miR-34a and miR-21 play roles in MNU-induced
breast carcinogenesis and the anticancer mechanism of flavonoids. Cervical cancer (CC) is one of the most maligt tumors and the second or third
most common type of cancer in women worldwide. The association between human
papillomavirus (HPV) and CC is widely known and accepted (99.7% of cases). At
present, the pathogenesis mechanisms of CC are not entirely clear. It has been
shown that inactivation of tumor suppressor genes and activation of oncogenes
play a significant role in carcinogenesis, caused by the genetic and epigenetic
alterations. In the past, it was generally thought that genetic mutation was a
key event of tumor pathogenesis, especially somatic mutation of tumor suppressor
genes. With deeper understanding of tumors in recent years, increasing evidence
has shown that epigenetic silencing of those genes, as a result of aberrant
hypermethylation of CpG islands in promoters and histone modification, is
essential to carcinogenesis and metastasis. The term epigenetics refers to
heritable changes in gene expression caused by regulation mechanisms, other than
changes in DNA sequence. Specific epigenetic processes include DNA methylation,
chromotin remodeling, histone modification, and microRNA regulations. These
alterations, in combination or individually, make it possible to establish the
methylation profiles, histone modification maps, and expression profiles
characteristic of this pathology, which become useful tools for screening, early
detection, or prognostic markers in cervical cancer. This paper reviews recent
epigenetics research progress in the CC study, and tries to depict the
relationships between CC and DNA methylation, histone modification, as well as
microRNA regulations. microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In
the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase
(GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218
expression was shown in primary breast tumors. Exogenous
miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein
level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a
positive correlation between miRNA-218 and GLCE mRNA, and negative correlation
between miRNA-218 and GLCE protein levels in breast tissues and primary tumors
in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional
regulation of GLCE in human breast tissue. A common scheme for the regulation of
GLCE expression in normal and tumor breast tissues is suggested. MicroRNAs, known as small noncoding MiRNAs, 19 to 24 nt in length, are important
gene regulators and recognized as key players in carcinogenesis. The mechanism
lies in that the MiRNAs can conjugate with their targeted mRNA and then lead to
the targeted mRNA degradation or repress their translation. Bioinformatic
analysis indicates that each MiRNA can regulate hundreds of gene targets and
could serve functionally as "oncogenes" or "tumor suppressor genes", and
therefore regulate multiple cellular processes relevant to carcinogenesis and
cancer progression. Up to now, there have been a lot of studies about the MiRNAs
which may play an important role in stomach neoplasms. The purpose of this paper
is to have a review of the present studies on the MiRNAs related to stomach
neoplasms, in order set basis for further study and their clinical application. Peroxisome-proliferator-activated receptors (PPARs) are ligand-activated nuclear
receptors that exert in the liver a transcriptional activity regulating a whole
spectrum of physiological functions, including cholesterol and bile acid
homeostasis, lipid/glucose metabolism, inflammatory responses, regenerative
mechanisms, and cell differentiation/proliferation. Dysregulations of the
expression, or activity, of specific PPAR isoforms in the liver are therefore
believed to represent critical mechanisms contributing to the development of
hepatic metabolic diseases, disorders induced by hepatic viral infections, and
hepatocellular adenoma and carcinoma. In this regard, specific PPAR agonists
have proven to be useful to treat these metabolic diseases, but for cancer
therapies, the use of PPAR agonists is still debated. Interestingly, in addition
to previously described mechanisms regulating PPARs expression and activity,
microRNAs are emerging as new important regulators of PPAR expression and
activity in pathophysiological conditions and therefore may represent future
therapeutic targets to treat hepatic metabolic disorders and cancers. Here, we
reviewed the current knowledge about the general roles of the different PPAR
isoforms in common chronic metabolic and infectious liver diseases, as well as
in the development of hepatic cancers. Recent works highlighting the regulation
of PPARs by microRNAs in both physiological and pathological situations with a
focus on the liver are also discussed. BACKGROUND: MicroRNA-101 (miR-101) expression is negatively associated with
tumor growth and blood vessel formation in several solid epithelial cancers.
However, the role of miR-101 in human breast cancer remains elusive.
RESULTS: MiR-101 was significantly decreased in different subtypes of human
breast cancer tissues compared with that in adjacent normal breast tissues
(P<0.01). Up-regulation of miR-101 inhibited cell proliferation, migration and
invasion, and promoted cell apoptosis in ER alpha-positive and ER alpha-negative
breast cancer cells and normal breast cells. Down-regulation of miR-101
displayed opposite effects on cell growth and metastasis. Further investigation
revealed a significant inverse correlation between the expression of miR-101 and
Stathmin1 (Stmn1), and miR-101 could bind to the 3'-untranslated region (UTR) of
Stmn1 to inhibit Stmn1 translation. The inhibition of cell growth and metastasis
induced by up-regulation of miR-101 was partially restored by overexpression of
Stmn1. Knockdown of Stmn1 attenuates the down-regulation of miR-101-mediated
enhancement of cell growth and metastasis. More importantly, in vivo analysis
found that Stmn1 mRNA and protein level in different subtypes of human breast
cancer tissues, contrary to the down-regulation of miR-101, were significantly
elevated.
CONCLUSIONS: This study demonstrates that down-regulation of miR-101 in
different subtypes of human breast cancer tissues is linked to the increase of
cellular proliferation and invasiveness via targeting Stmn1, which highlights
novel regulatory mechanism in breast cancer and may provide valuable clues for
the future clinical diagnosis of breast cancer. Considerable attention and an enormous amount of resources have been dedicated
to cancer biomarker discovery and validation. However, there are still a limited
number of useful biomarkers available for clinical use. An ideal biomarker
should be easily assayed with minimally invasive medical procedures but possess
high sensitivity and specificity. Commonly used circulating biomarkers are
proteins in serum, most of which require labor-intensive analysis hindered by
low sensitivity in early tumor detection. Since the deregulation of microRNA
(miRNA) is associated with cancer development and progression, profiling of
circulating miRNAs has been used in a number of studies to identify novel
minimally invasive miRNA biomarkers. In this review, we discuss the origin of
the circulating cell-free miRNAs and their carriers in blood. We summarize the
clinical use and function of potentially promising miRNA biomarkers in a variety
of different cancers, along with their downstream target genes in tumor
initiation and development. Additionally, we analyze some technical challenges
in applying miRNA biomarkers to clinical practice. Aberrant activation of the Wnt/β-catenin signal pathway is frequently observed
in hepatocellular carcinoma (HCC). β-Catenin is the major cellular effector of
Wnt signaling and inactivation of adenomatous polyposis coli (APC) results in
nuclear accumulation of β-catenin. Therefore, it was speculated that APC
inhibition could play important roles in activating the Wnt/β-catenin pathway
and in HCC progression. In this study, we report that miR-106b expression is
markedly upregulated in hepatoma cells and hepatoma tissues compared with
immortalized normal liver epithelial cells and normal hepatic tissues. Ectopic
expression of miR-106b induces the proliferation and anchorage-independent
growth of hepatoma cells, whereas inhibition of miR-106b reduced this effect.
Furthermore, miR-106b upregulation in hepatoma cells modulated entry into the
G(1)/S transitional phase by upregulating cyclin D1 and downregulating APC.
Moreover, we demonstrated that miR-106b downregulates APC expression by directly
targeting the 3'-untranslated region of APC messenger RNA. Taken together, our
results suggest that miR-106b plays an important role in promoting the
proliferation of human hepatoma cells and presents a novel mechanism of micro
RNA-mediated direct suppression of APC expression in cancer cells. Nickel (Ni) compounds are well-recognized human carcinogens, yet the molecular
mechanisms by which they cause human cancer are still not well understood.
MicroRNAs (miRNAs), which are small non-coding RNAs, are involved in diverse
biological functions and carcinogenesis. In previous study, we identified
upregulation of DNA methyltransferase 1 (DNMT1) expression in nickel sulfide
(NiS)-transformed human bronchial epithelial (16HBE) cells. Here, we
investigated whether some miRNAs are aberrantly expressed and targets DNMT1 in
NiS-transformed cells. Our results showed that the expression of miRNA-152
(miR-152) was specifically downregulated in NiS-transformed cells via promoter
DNA hypermethylation, whereas ectopic expression of miR-152 in NiS-transformed
cells resulted in a marked reduction of DNMT1 expression. Further experiments
revealed that miR-152 directly downregulated DNMT1 expression by targeting the
3' untranslated regions of its transcript. Interestingly, treatment of DNMT
inhibitor, 5-aza-2-deoxycytidine, or depletion of DNMT1 led to increased miR-152
expression by reversion of promoter hypermethylation, DNMT1 and MeCP2 binding to
miR-152 promoter in NiS-transformed cells. Moreover, inhibition of miR-152
expression in 16HBE cells could increase DNMT1 expression and result in an
increase in DNA methylation, DNMT1 and MeCP2 binding to miR-152 promoter,
indicating an interaction between miR-152 and DNMT1 is regulated by a
double-negative circuit. Furthermore, ectopic expression of miR-152 in
NiS-transformed cells led to a significant decrease of cell growth. Conversely,
inhibition of miR-152 expression in 16HBE cells significantly increased cell
growth. Taken together, these observations demonstrate a crucial functional
crosstalk between miR-152 and the DNMT1 via a feedback loop involved in
NiS-induced maligt transformation. MicroRNAs (miRNAs) are a class of non-coding RNA, which have recently been shown
to have a wide variety of regulatory functions in relation to gene expression.
Since their identification nearly 20 years ago, miRNAs have been found to play
an important role in cancer, including in neurofibromatosis type 1
(NF1)-associated tumours. NF1 is the most commonly inherited tumour
predisposition syndrome and can lead to maligcy via the development of
maligt peripheral nerve sheath tumours (MPNSTs). Although the mechanisms by
which benign neurofibromas develop into MPNSTs still remain to be elucidated, it
is becoming increasingly clear that miRNAs play a key role in this process and
have the potential to be used as both diagnostic and prognostic markers of
tumorigenesis. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression by
modulating numerous target mRNAs expression at posttranscriptional level.
Extensive studies have shown that miRNAs are critical in various important
biological processes, including cell growth, proliferation, differentiation,
development, and apoptosis. In terms of their importance, miRNA dysfunction has
been associated with a broad range of diseases. Increased number of studies have
shown that miRNAs can functionally interact with a wide spectrum of
environmental factors (EFs) including drugs, industrial materials, virus and
bacterial pathogens, cigarette smoking, alcohol, nutrition, sleep, exercise,
stress, and radiation. More importantly, the interactions between miRNAs and EFs
have been shown to play critical roles in determining abnormal phenotypes and
diseases. In this paper, we propose an outline of the current knowledge about
specific roles of miRNAs in their interactions with various EFs and analyze the
literatures detailing miRNAs-EFs interactions in the context of various of
diseases. MicroRNAs (miRNAs or miR) have been integrated into tumorigenic programs as
either oncogenes or tumor suppressor genes. The miR-124 was reported to be
attenuated in several tumors, such as glioma, medulloblastoma and hepatocellular
carcinoma. However, its role in cancer remains greatly elusive. In this study,
we show that the miR-124 expression is significantly suppressed in human breast
cancer specimens, which is reversely correlated to histological grade of the
cancer. More intriguingly, ectopic expression of miR-124 in aggressive breast
cancer cell lines MDA-MB-231 and BT-549 strongly inhibits cell motility and
invasive capacity, as well as the epithelial-mesenchymal transition process.
Also, lentivirus-delivered miR-124 endows MDA-MB-231 cells with the ability to
suppress cell colony formation in vitro and pulmonary metastasis in vivo.
Further studies have identified the E-cadherin transcription repressor Slug as a
direct target gene of miR-124; its downregulation by miR-124 increases the
expression of E-cadherin, a hallmark of epithelial cells and a repressor of cell
invasion and metastasis. Moreover, knockdown of Slug notably impairs the
motility of MDA-MB-231 cells, whereas re-expression of Slug abrogates the
reduction of motility and invasion ability induced by miR-124 in MDA-MB-231
cells. These findings highlight an important role for miR-124 in the regulation
of invasive and metastatic potential of breast cancer and suggest a potential
application of miR-124 in cancer treatment. The aim of this study was to evaluate the effects and molecular mechanisms of
everolimus on Panc-1 human pancreatic cancer cells. Panc-1 human pancreatic
cancer cells were treated with everolimus (10 μg/ml) at selected time points (6,
12 and 24 h). Cell proliferation and apoptosis were evaluated by MTT and flow
cytometric analyses. The glycolytic activity was determined by measuring the
activity of the key enzyme lactate dehydrogenase (LDH) and lactate production.
The activity of mammalian target of rapamycin (mTOR) signaling was measured by
western blotting. The expression of genes, including hexokinase 2 (HK2) and
microRNA-143 (miR-143), was evaluated by real-time polymerase chain reaction
(PCR). The administration of everolimus time-dependently inhibited proliferation
and glycolysis and induced apoptosis in the Panc-1 human pancreatic cancer
cells. As the time of treatment with everolimus increased, the mTOR signaling
activity decreased, indicated by lower phosphorylation levels of S6 kinase;
however, the phosphorylation levels of mTOR barely changed. Moreover, our data
showed an everolimus-induced increase in miR-143 and decrease in HK2 in Panc-1
cells in a time-dependent manner. In conclusion, the current study indicates a
novel role of everolimus in its antitumor effect as an inhibitor of glycolysis
in Panc-1 human pancreatic cancer cells. Furthermore, our data highlights the
significance of exploring the mechanisms of everolimus and miR-143 in maligt
tumors. Liver cancer (hepatocellular carcinoma, HCC) is a major maligcy worldwide.
Etiologically, hepatocarcinogenesis is closely associated with HBV and HCV
infections; however, its underlying molecular mechanism is not completely
understood. MicroRNAs are a class of small non-coding RNAs that negatively
regulate gene expression by interacting with the 3'UTR of protein-coding mRNA.
MicroRNAs are implicated in nearly all major biological and cellular events, and
recent findings further link microRNA deregulation to human carcinogenesis. In
this review, we will focus on the aberrant expression of miRNAs in liver cancer
and the pathological implications and molecular functions of some
well-characterized oncogenic and tumor suppressive miRNAs. Finally, the clinical
prospect of miRNAs as a novel diagnostic and therapeutic intervention will be
discussed. BACKGROUND: MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding
RNA molecules whose deregulation of expression can contribute to human disease
including the multistep processes of carcinogenesis in human. Circulating miRNAs
are emerging biomarkers in many diseases and cancers such as type 2 diabetes,
pulmonary disease, colorectal cancer, and gastric cancer among others; however,
defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that
could serve as a biomarker for diagnosis or in the follow-up has not been done
yet.
METHODS: TaqMan miRNA microarray was performed to identify deregulated miRNAs in
the plasma of AML patients. Quantitative real-time RT-PCR was used to validate
the results. Receiver-operator characteristic (ROC) curve analysis was conducted
to evaluate the diagnostic accuracy of the highly and significantly identified
deregulated miRNA(s) as potential candidate biomarker(s).
RESULTS: The plasma expression level of let-7d, miR-150, miR-339, and miR-342
was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the
AML group at diagnosis compared to healthy controls. ROC curve analyses revealed
an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119- 0.9581;
P<0.0001) and 0.8125 (95% CI: 0.6796-0.9454; P=0.0005) for miR-150, and miR-342
respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated
AUC of 0.86 (95% CI: 0.7819-0.94; P<0.0001) indicating the additive effect in
the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the
expression level of these two miRs in complete remission AML patients resembled
that of healthy controls.
CONCLUSIONS: Our findings indicated that plasma miR-150 and miR-342 are novel
important promising biomarkers in the diagnosis of AML. These novel and
promising markers warrant validation in larger prospective studies. The eukaryotic complexity involves the expression and regulation of genes via
RNA-DNA, RNA-RNA, DNA-protein and RNA-protein interactions. Recently, the role
of RNA molecules in the regulation of genes in higher organisms has become more
evident, especially with the discovery that about 97% of the transcriptional
output in higher organisms are represented as noncoding RNAs: rRNA, snoRNAs,
tRNA, transposable elements, 5' and 3' untranslated regions, introns, intergenic
regions and microRNAs. MicroRNAs function by negatively regulating gene
expression via degradation or translational inhibition of their target mRNAs and
thus participate in a wide variety of physiological and pathological cellular
processes including: development, cell proliferation, differentiation, and
apoptosis pathways. MicroRNA expression profiles in many types of cancers have
been identified. Recent reports have revealed that the expression profiles of
microRNAs change in various human cancers and appear to function as oncogenes or
tumor suppressors. Abnormal microRNA expression has increasingly become a common
feature of human cancers. In this review, we summarize the latest progress on
the involvement of microRNAs in different types of cancer and their potential
use as potential diagnostic and prognostic tumor biomarkers in the future. The interplay between tumor cells and their microenvironment plays a pivotal
role in tumor development and progression. Although a growing body of evidence
has established the importance of the tumor microenvironment, an understanding
of the crosstalk between its components and cancer cells remains elusive. The
pathways triggered by microenvironmental factors could modulate cancer-related
gene transcription, also affecting small noncoding RNAs, microRNAs, which have
emerged as key posttranscriptional regulators of gene expression, directly
involved in human cancers. Although microRNAs regulate most biological
mechanisms, their role in the tumor microenvironment has only recently become
the focus of intense research. In this paper, we focus on the intertwined
connection between the tumor microenvironment and aberrant expression of
microRNAs involved in carcinogenesis. We also discuss the emerging roles of
microRNAs in the tumor microenvironment as it relates to cancer progression. We
conclude that microRNAs are critical for our understanding of the development of
cancer, and that targeting microRNA signaling pathways in the microenvironment
as well as in tumor cells opens new therapeutic avenues to the global control of
cancer. |
Which type of GTPases is required for amino acid-dependent activation of mTORC1? | Heterodimeric Rag GTPases are required for amino-acid-mediated mTORC1 activation at the lysosome | Amino acids are required for activation of the mammalian target of rapamycin
(mTOR) kinase, which regulates protein translation, cell size, and autophagy.
However, the amino acid sensor that directly couples intracellular amino
acid-mediated signaling to mTORC1 is unknown. Here we show that leucyl-tRNA
synthetase (LRS) plays a critical role in amino acid-induced mTORC1 activation
by sensing intracellular leucine concentration and initiating molecular events
leading to mTORC1 activation. Mutation of LRS amino acid residues important for
leucine binding renders the mTORC1 pathway insensitive to intracellular levels
of amino acids. We show that LRS directly binds to Rag GTPase, the mediator of
amino acid signaling to mTORC1, in an amino acid-dependent manner and functions
as a GTPase-activating protein (GAP) for Rag GTPase to activate mTORC1. This
work demonstrates that LRS is a key mediator for amino acid signaling to mTORC1. mTORC1 (mammalian target of rampamycin complex 1) is a highly conserved protein
complex regulating cell growth and metabolism via its kinase mTOR (mammalian
target of rapamycin). The activity of mTOR is under the control of various
GTPases, of which Rheb and the Rags play a central role. The presence of amino
acids is a strict requirement for mTORC1 activity. The heterodimeric Rag GTPases
localize mTORC1 to lysosomes by their amino-acid-dependent interaction with the
lysosomal Ragulator complex. Rheb is also thought to reside on lysosomes to
activate mTORC1. Rheb is responsive to growth factors, but, in conjunction with
PLD1 (phospholipase D1), is also an integral part of the machinery that
stimulates mTORC1 in response to amino acids. In the present article, we provide
a brief overview of novel mechanisms by which amino acids affect the function of
Rags. On the basis of existing literature, we postulate that Rheb is activated
at the Golgi from where it will travel to lysosomes. Maturation of endosomes
into lysosomes may be required to assure a continuous supply of GTP-bound Rheb
for mTORC1 activation, which may help to drive the maturation process. Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is
mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in
an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP,
the immediate activator. Here we examine whether the ability of amino acids to
regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of
Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid,
spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP.
Mutation of the Rag P-loop corresponding to Ras(Ser-17) abolishes guanyl
nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or
RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type
and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with
transient expression, but binding to mTORC1 in vitro is entirely independent of
Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or
P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite
amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid
withdrawal as in parental cells. Rag heterodimers extracted from (32)P-labeled
whole cells, or just from the pool associated with the lysosomal membrane,
exhibit constitutive [(32)P]GTP charging that is unaltered by amino acid
withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP
charging. Raptor binding to Rag, although necessary, is not sufficient for
mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to
Rheb-GTP. The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of
cellular and organismal growth, and hyperactivation of this pathway is
implicated in the pathogenesis of many human diseases including cancer and
diabetes. mTORC1 promotes growth in response to the availability of nutrients,
such as amino acids, which drive mTORC1 to the lysosomal surface, its site of
activation. How amino acid levels are communicated to mTORC1 is only recently
coming to light by the discovery of a lysosome-based signaling system composed
of Rags (Ras-related GTPases) and Ragulator v-ATPase, GATOR (GAP activity
towards Rags), and folliculin (FLCN) complexes. Increased understanding of this
pathway will not only provide insight into growth control but also into the
human pathologies triggered by its deregulation. Author information:
(1)Whitehead Institute for Biomedical Research and Massachusetts Institute of
Technology, Department of Biology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA. Koch Institute for Integrative Cancer
Research, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Broad Institute of
Harvard and Massachusetts Institute of Technology, 7 Cambridge Center,
Cambridge, MA 02142, USA.
(2)Harvard Medical School, 260 Longwood Avenue, Boston, MA 02115, USA.
(3)Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical
School, 220 Longwood Avenue, Boston, MA 02115, USA.
(4)Whitehead Institute for Biomedical Research and Massachusetts Institute of
Technology, Department of Biology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA. Koch Institute for Integrative Cancer
Research, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Broad Institute of
Harvard and Massachusetts Institute of Technology, 7 Cambridge Center,
Cambridge, MA 02142, USA. [email protected]. The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates
environmental and intracellular signals to regulate cell growth. Amino acids
stimulate mTORC1 activation at the lysosome in a manner thought to be dependent
on the Rag small guanosine triphosphatases (GTPases), the Ragulator complex, and
the vacuolar H(+)-adenosine triphosphatase (v-ATPase). We report that leucine
and glutamine stimulate mTORC1 by Rag GTPase-dependent and -independent
mechanisms, respectively. Glutamine promoted mTORC1 translocation to the
lysosome in RagA and RagB knockout cells and required the v-ATPase but not the
Ragulator. Furthermore, we identified the adenosine diphosphate ribosylation
factor-1 GTPase to be required for mTORC1 activation and lysosomal localization
by glutamine. Our results uncover a signaling cascade to mTORC1 activation
independent of the Rag GTPases and suggest that mTORC1 is differentially
regulated by specific amino acids. |
In which genomic regions are Alu enriched? | There are regions such as the four homeobox gene clusters, which are nearly devoid of these repeats that contrast with repeat dense regions in other transcriptionally active regions of the genome. Alu elements are more clustered in genes which are involved in metabolism, transport, and signaling processes. In contrast, they are significantly fewer in genes coding for information pathway components as well as structural proteins.
This bias in Alu distribution is independent of the effect of Alu density of the flanking genomic region and is also not affected by the GC content of the gene and its upstream and downstream regions. | Cloned DNA fragments form the human beta-like globin genomic region can be
transcribed in vitro by RNA polymerase III. We have investigated the structure
of two templates and their transcripts by DNA sequencing, size fractionation of
ribonuclease T1 generated oligonucleotides, and ribonuclease H digestion of RNA
: DNA duplexes. The data indicate the repetitive DNA sequences, members of the
Alu family of interpersed 300 bp reiterated DNA, are imbedded in both templates.
The RNAs transcribed from them are composed of an entire Alu family sequence at
their 5' ends linked to 3' ends of non-repetitive sequence. Alu elements belonging to the previously identified "young" subfamilies are
thought to have inserted in the human genome after the divergence of humans from
non-human primates and therefore should not be present in non-human primate
genomes. Polymerase chain reaction (PCR) based screening of over 500 Alu
insertion loci resulted in the recovery of a few "young" Alu elements that also
resided at orthologous positions in non-human primate genomes. Sequence analysis
demonstrated these "young" Alu insertions represented gene conversion events of
pre-existing ancient Alu elements or independent parallel insertions of older
Alu elements in the same genomic region. The level of gene conversion between
Alu elements suggests that it may have a significant influence on the single
nucleotide diversity within the genome. All the instances of multiple
independent Alu insertions within the same small genomic regions were recovered
from the owl monkey genome, indicating a higher Alu amplification rate in owl
monkeys relative to many other primates. This study suggests that the majority
of Alu insertions in primate genomes are the products of unique evolutionary
events. A comprehensive analysis of two Alu Y lineage subfamilies was undertaken to
assess Alu-associated genomic diversity and identify new Alu insertion
polymorphisms for the study of human population genetics. Recently integrated
Alu elements (283) from the Yg6 and Yi6 subfamilies were analyzed by polymerase
chain reaction (PCR), and 25 of the loci analyzed were polymorphic for insertion
presence/absence within the genomes of a diverse array of human populations.
These newly identified Alu insertion polymorphisms will be useful tools for the
study of human genomic diversity. Our screening of the Alu insertion loci also
resulted in the recovery of several "young" Alu elements that resided at
orthologous positions in nonhuman primate genomes. Sequence analysis
demonstrated these "young" Alu insertions were the products of gene conversion
events of older, preexisting Alu elements or independent parallel forward
insertions of older Alu elements in the same short genomic region. The level of
gene conversion between Alu elements suggests that it may have an influence on
the single nucleotide polymorphism within Alu elements in the genome. We have
also identified two genomic deletions associated with the retroposition and
insertion of Alu Y lineage elements into the human genome. This type of Alu
retroposition-mediated genomic deletion is a novel source of lineage-specific
evolution within primate genomes. Pathogenic Alu element insertions are rarely reported, whereas their occurrence
is expected to be much higher. Alu containing alleles are usually out-competed
during the PCR process and consequently undetectable with the classical
screening methods. However, with the introduction of the next generation
sequencing (NGS) technology in the diagnostic field, new opportunities are
emerging. NGS data for a particular genomic region can be seen as the summation
of all the individual sequences (reads) obtained for that region and no longer
as the mean of this sum as it is the case for traditional Sanger sequencing.
Because each single read covering that region is expected to be generated from a
different template molecule, the presence of one single mutant read must
theoretically be sufficient to identify the mutation. However, generation and
identification of mutant reads bearing Alu insertions remains challenging and
several wet/dry bench parameters need to be optimized. Hereby we present the
proof of principle of a NGS-based mutation screening procedure allowing the
detection of inherited Alu insertions within any predefined sequence by
investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2. |
What is the synonym of the lubag disease? | Lubag disease is also known as X-linked dystonia-parkinsonism (XDP). This disease is characterized by dystonia and parkinsonism, and afflicts Filipino men, and rarely, women originating principally from the Panay Island. | We report a patient with Lubag (X-linked dystonia-parkinsonism) who presented
with severe respiratory stridor from adductor laryngeal breathing dystonia.
Emergency tracheostomy was necessary, and subsequent laryngeal injection with
botulinum toxin led to worsening aspiration. Botulinum toxin injection for
severe lingual dystonia was successful. Regional and global metabolic rates for glucose (rCMRGlc and GMR) were estimated
using [18F]fluorodeoxyglucose and positron emission tomography in 3 patients
with Filipino X-linked dystonia-parkinsonism (lubag). In all 3 patients a
selective reduction in normalized striatal glucose metabolism (rCMRGlc/GMR) was
observed compared with 15 normal volunteer subjects. Presynaptic nigrostriatal
function was assessed in these patients using [18F]fluorodopa and positron
emission tomography. Striatal rate constants for [18F]flurodopa uptake were
found to be in the normal range in all 3 patients with lubag. These findings
suggest that the extrapyramidal manifestations of lubag are metabolically
localized to the striatum and that clinical parkinsonism in these patients may
be secondary to extranigral factors. Lubag is an x-linked recessive dystonia parkinsonism that affects Filipino men
originating principally from the Panay Island. Linkage analysis has confirmed
the mode of inheritance and localized the disease gene to the proximal long arm
of the x-chromosome. We studied the brain of a 34 year old Filipino man affected
with lubag. He developed truncal dystonia at age 30, which subsequently
generalized. With disease progression, he also presented with parkinsonism
including, rigidity, bradykinesia, and impaired balance. His symptoms were
largely unaffected by medication and, at age 34, he underwent a right
cryothalamotomy. He died suddenly 2 days after the procedure. The principal
neuropathological findings were neuronal loss and a multifocal mosaic pattern of
astrocytosis restricted to the caudate and lateral putamen. Similar findings
have been reported in two other men with dystonia--one Filipino and the other
non-Filipino. The similar pathology of the two Filipino men suggests that this
is the pathology of lubag. Recognition of this pathology in a non-Filipino man
suggests that the mutation causing lubag may not be restricted to the Filipino
population. X-linked dystonia-parkinsonism (XDP), or Lubag syndrome, is known to cause
progressive dystonia, with or without parkinsonism, among Filipino male adults
with maternal roots from the Philippine island of Panay. We present
cinematographic material of 11 cases of Lubag carrying the XDP haplotypes who
manifest with a wide spectrum of movement disorders, including dystonia, tremor,
parkinsonism, myoclonus, chorea, and myorhythmia. Because of overlapping
features, Lubag patients are commonly misdiagnosed as idiopathic dystonia,
essential tremor, Parkinson's disease, or Parkinson's-plus syndromes. Thus, it
is imperative to elicit an exhaustive family history in any Filipino male adult
who presents with a movement disorder. The clinico-pathological study of a new type of familial parkinsonism with
striatal degeneration is reported. The inheritance mode was autosomal recessive,
and three out of four offspring of married cousins developed parkinsonism in
their early adulthood. Their clinical signs were rigidity, bradykinesia,
postural instability and dysarthria. These symptoms were slowly progressive and
responsive to levodopa therapy to a variable degree. On cerebral magnetic
resoce imaging, T2 and proton density-weighted images showed hyperintensity
in the bilateral putamina. The neuropathological study of one case revealed
atrophy of the bilateral putamina and caudate nuclei, and a severe neuronal loss
and gliosis in the putamina. Patchy mosaicism of normal and degenerated tissue
was observed in the putamina. A similar mode of the degeneration was mildly seen
in the caudate nuclei. The substantia nigra showed atrophy of the pars
reticulata, and mild to moderate neuronal loss of the pars compacta with rostral
domice, but no Lewy bodies were observed. These neuropathological findings
differed from those of Parkinson's disease or juvenile parkinsonism, but mimic
to those of X-linked dystonia parkinsonism (Lubag). It seems that this familial
bilateral striatal degeneration is a new variant of familial parkinsonism. We report on an Italian family in which three brothers and their maternal
grandfather had a generalized early-onset dystonia with mild parkinsonian signs.
Genetic testing excluded the rapid-onset dystonia-parkinsonism locus (DYT12;
OMIM*128235), autosomal recessive Parkin locus (PARK2; OMIM *602544), and DYT1
dystonia. Three affected siblings were found to share an identical haplotype at
the X-linked dystonia-parkinsonism locus (XDP; Lubag; OMIM*314250). This
haplotype differed from the haplotype observed in Filipino patients, ruling out
the hypothesis of a common underlying mutation. In addition, direct sequencing
analysis of the putative disease causing changes observed in Filipino patients
were not found in the Italian patients. The condition we describe could be a
newly recognized dystonia syndrome with parkinsonism. "Lubag" or X-linked dystonia-parkinsonism (XDP) is a genetic syndrome afflicting
Filipino men. Intracranial surgical procedures for Lubag have been unsuccessful.
We report a 45-year-old Filipino male with genetically confirmed XDP who
underwent bilateral pallidal deep brain stimulation (DBS) surgery. The patient
started to exhibit improvement on initial programming, most notably of his
severe jaw-opening dystonia. At 1-year follow-up, his Burke-Fahn-Marsden
dystonia score and motor Unified Parkinson's Disease Rating Scale score were
improved by 71% and 62%, respectively, with the stimulators on compared to
stimulators off state. Bilateral pallidal DBS may be a viable option for Lubag
patients with medically refractory symptoms. Sex-linked dystonia parkinsonism (XDP, DYT3, "Lubag") is an adult-onset,
progressive, debilitating movement disorder first described in Filipino males
from Panay Islands in 1975. XDP manifests predomitly as torsion dystonia,
later combined with or sometimes replaced with parkinsonism. Within the Island
of Panay, the prevalence rate is highest in the province of Capiz, where 1:4000
men suffer from the disorder. There is a high degree of penetrance and
generalization. While women often serve as carriers, XDP is not limited to men.
An updated XDP Philippine registry (as of January 2010) has identified 505
cases, with 500 males and 5 females. While some report that females may carry a
milder form of the disorder, in our experience, both sexes generally follow a
similar progressive clinical course. Lubag disease is a genetic X-linked dystonia-parkinsonism syndrome afflicting
Filipino men. This disease is characterized by dystonia dominating the first
10-15 years of the disorder, which is associated with or replaced by
parkinsonian features in later years of life. A 49-year-old man with Lubag
disease underwent general anesthesia for deep brain stimulation (DBS) surgery.
Anesthesia was maintained mainly with propofol, remifentanil, rocuronium
bromide, and sevoflurane. During magnetic resoce imaging, the patient was
anesthetized with midazolam, fentanyl, and rocuronium bromide. The surgery was
completed safely using these anesthetic agents. After DBS, some symptoms
including involuntary movement improved within 10 days. |
Dracorhodin perchlorate was tested for treatment of which cancers? | Dracorhodin perchlorate induce apoptosis in prostate cancer, gastric tumor, melanoma and premyelocytic leukemia. | Dracorhodin perchlorate inhibited proliferation of several tumor cell lines. The
drug induced oligonucleosomal fragmentation of DNA in HeLa cells and increased
caspase-3, -8, -9 activities followed by the degradation of caspase-3
substrates, inhibitor of caspase-dependent DNase, and poly-(ADP-ribose)
polymerase. It also increased caspase-1 activity and a caspase-1 inhibitor,
Ac-YVAD-cmk, and a caspase-10 inhibitor z-AEVD-fmk, also reduced
dracorhodin-perchlorate-induced HeLa cell death. Dracorhodin perchlorate
decreased the expression of anti-apoptotic mitochondrial protein, Bcl-X(L), but
not Bcl-2; and it increased the expression of pro-apoptotic protein, Bax.
Dracorhodin perchlorate induced a sustained generation of reactive oxygen
species (ROS) in HeLa cells; caspase-1 inhibitor, Ac-YVAD-cmk, and caspase-3
inhibitor, z-DEVD-fmk, attenuated the generation of ROS. Taken together, our
results indicate that dracorhodin perchlorate alters the intracellular redox
status, changed the balance of Bcl-X(L) and Bax protein expression, and induces
apoptosis through caspase pathways in HeLa cells. Dracorhodin perchlorate, an anthocyanin red pigment, induces human melanoma
A375-S2 cell death through the apoptotic pathway. Caspase-3, -8, -9, and -10
inhibitors partially reversed the cell death induced by dracorhodin perchlorate.
Caspase-3 and -8 were activated, followed by the degradation of caspase-3
substrates, the inhibitor of caspase-activated DNase, and poly-(ADP-ribose)
polymerase. Dracorhodin perchlorate upregulated the expression ratio of
Bax/Bcl-2 and significantly increased the expression of p53 and p21(WAF1)
proteins. The cell death was partially reduced by the mitogen-activated protein
kinase c-JUN NH2-terminal protein kinase (JNK MAPK) inhibitor (SP600125) and p38
MAPK inhibitor (SB 203580), while the MEK inhibitor (PD98059) augmented cell
death; the drug induced sustained phosphorylation of JNK and p38 MAPK. Moreover,
the Fas agonistic antibody CH-11 has a synergistic effect with dracorhodin
perchlorate. The phoshatidylinositol 3-kinase (PI3-K) family inhibitor wortmanin
and tyrosine kinase inhibitor genistein rescued the viability loss induced by
dracohodin perchlorate. Taken together, dracorhodin perchlorate induces
apoptosis in A375-S2 cells via accumulation of p53, alters the Bax/Bcl-2 ratio,
and activates caspases and p38/JNK MAPKs. Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic
leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and
-10 inhibitors partially reversed the cell death induced by dracorhodin
perchlorate. Caspase-3 and -8 were activated followed to the degradation of
caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and
poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the
expression ratio of mitochondrial proteins, Bax/Bcl-XL. The cell death was
accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced
by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor
(SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60
cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs. The growth inhibition and pro-apoptosis effects of dracorhodin perchlorate on
human prostate cancer PC-3 cell line were examined. After administration of
10-80 μmol/L dracorhodin perchlorate for 12-48 h, cell viability of PC-3 cells
was measured by MTT colorimetry. Cell proliferation ability was detected by
colony formation assay. Cellular apoptosis was inspected by acridine
orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent
staining, and flow cytometry (FCM) with annexin V-FITC/propidium iodide dual
staining. The results showed that dracorhodin perchlorate inhibited the growth
of PC-3 in a dose- and time-dependent manner. IC50 of dracorhodin perchlorate on
PC-3 cells at 24 h was 40.18 μmol/L. Cell clone formation rate was decreased by
86% after treatment with 20 μmol/L of dracorhodin perchlorate. Some cells
presented the characteristic apoptotic changes. The cellular apoptotic rates
induced by 10-40 μmol/L dracorhodin perchlorate for 24 h were 8.43% to 47.71%
respectively. It was concluded that dracorhodin perchlorate significantly
inhibited the growth of PC-3 cells by suppressing proliferation and inducing
apoptosis of the cells. Dracorhodin perchlorate has been recently shown to induce apoptotic cell death
in cancer cells. However, the molecular mechanisms underlying these effects are
unknown in human gastric tumor cells. In this study, effects of Dracorhodin
perchlorate on cell viability, cell cycle, and apoptosis were investigated in
SGC-7901 cells. The results showed that Dracorhodin perchlorate induced cellular
and DNA morphological changes and decreased the viability of SGC-7901 cells.
Dracorhodin perchlorate-mediated cell cycle arrest was associated with a marked
decrease in protein levels of phosphorylated retinoblastoma and E2F1.
Dracorhodin perchlorate-induced apoptosis is mediated via upregulation of p53,
inhibiting the activation of PI3K/Akt, and NF-κB, thereby decreasing the
expression of the anti-apoptotic proteins, Bcl-2 and Bcl-XL. Interestingly, we
also found that Dracorhodin perchlorate significantly suppressed the
IGF-1-induced phosphorylation of Akt in the stably expressing EGFP-Akt
recombit CHO-hIR cells and inhibited TNF-induced NF-κB transcriptional
activity in the NF-κBp65-EGFP recombit U2OS cells, indicating that inhibition
of PI3K/Akt and NF-κB may provide a molecular basis for the ability of
Dracorhodin perchlorate to induce apoptosis. Dracorhodin perchlorate induced
up-regulation of p53, thereby resulting in the activation of its downstream
targets p21 and Bax following the dissipation of mitochondrial membrane
potential and activation of caspase-3 and its substrate, PARP. Moreover,
Dracorhodin perchlorate dramatically enhanced the wortmannin- and TNF-induced
apoptosis in SGC-7901 cells. These results reveal functional interplay among the
PI3K/Akt, p53 and NF-κB pathways that are frequently deregulated in cancer and
suggest that their simultaneous targeting by Dracorhodin perchlorate could
result in efficacious and selective killing of cancer cells. OBJECTIVE: Dracorhodin perchlorate (DP) was a synthetic analogue of the
antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could
induce apoptosis in human prostate cancer, human gastric tumor cells and human
melanoma, but the cytotoxic effect of DP on human breast cancer was not
investigated. This study would investigate whether DP was a candidate chemical
of anti-human breast cancer.
METHODS: The MTT assay reflected the number of viable cells through measuring
the activity of cellular enzymes. Phase contrast microscopy visualized cell
morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst
33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine
123 staining was used to detect cell apoptosis and mitochondrial membrane
potential (MMP). Real time PCR detected mRNA level. Western blot examined
protein expression.
RESULTS: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP
inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of
Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and
DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing
factor (AIF) and cytochrome c which localized in mitochondrial in physiological
condition were released into cytoplasm when MMP was decreased. DP activated
caspase-9, which was the downstream of mitochondrial pathway. Therefore DP
decreased MMP to release AIF and cytochrome c into cytoplasm, further activating
caspase 9, lastly led to apoptosis.
CONCLUSION: Therefore DP was a candidate for anti-breast cancer, DP induced
apoptosis of MCF-7 through mitochondrial pathway. |
What is the role of venous angioplasty in multiple sclerosis? | Chronic cerebrospinal venous insufficiency (CCSVI) may be an important factor in the pathogenesis of multiple sclerosis (MS). The proposed treatment for CCSVI is percutaneous transluminal angioplasty, also known as the 'liberation procedure', which is claimed to improve the blood flow in the brain, thereby alleviating some of the symptoms of MS. There have been reports to suggest that venous angioplasty in MS patients is a feasible and safe procedure and is associated with improved disease symptom severity, quality of life, and corrects blood pressure deviation, improves CSF dynamics. Open venous reconstruction of the internal jugular vein has also been tried with good results.
However, some authors have failed to document beneficial value of venous angioplasty in MS patients and systematic review has suggested that there is currently no high level evidence to support or refute the efficacy or safety of percutaneous transluminal angioplasty for treatment of CCSVI in people with MS. | OBJECTIVE: Chronic cerebrospinal venous insufficiency (CCSVI) is characterized
by combined stenoses of the principal pathways of extracranial venous drainage,
including the internal jugular veins (IJVs) and the azygous (AZY) vein, with
development of collateral circles and insufficient drainage shown by increased
mean transit time in cerebral magnetic resoce (MR) perfusion studies. CCSVI
is strongly associated with multiple sclerosis (MS). This study evaluated the
safety of CCSVI endovascular treatment and its influence on the clinical outcome
of the associated MS.
METHODS: Sixty-five consecutive patients with CCSVI, subdivided by MS clinical
course into 35 with relapsing remitting (RR), 20 with secondary progressive
(SP), and 10 with primary progressive (PP) MS, underwent percutaneous
transluminal angioplasty (PTA). Mean follow-up was 18 months. Vascular outcome
measures were postoperative complications, venous pressure, and patency rate.
Neurologic outcome measures were cognitive and motor function assessment, rate
of MS relapse, rate of MR active positive-enhanced gadolinium MS lesions (Gad+),
and quality of life (QOL) MS questionnaire.
RESULTS: Outpatient endovascular treatment of CCSVI was feasible, with a minor
and negligible complication rate. Postoperative venous pressure was
significantly lower in the IJVs and AZY (P < .001). The risk of restenosis was
higher in the IJVs compared with the AZY (patency rate: IJV, 53%; AZY, 96%; odds
ratio, 16; 95% confidence interval, 3.5-72.5; P < .0001). CCSVI endovascular
treatment significantly improved MS clinical outcome measures, especially in the
RR group: the rate of relapse-free patients changed from 27% to 50%
postoperatively (P < .001) and of MR Gad+ lesions from 50% to 12% (P < .0001).
The Multiple Sclerosis Functional Composite at 1 year improved significantly in
RR patients (P < .008) but not in PP or SP. Physical QOL improved significantly
in RR (P < .01) and in PP patients (P < .03), with a positive trend in SP (P <
.08). Mental QOL showed significant improvement in RR (P < .003) and in PP (P <
.01), but not in SP.
CONCLUSIONS: PTA of venous strictures in patients with CCSVI is safe, and
especially in patients with RR, the clinical course positively influenced
clinical and QOL parameters of the associated MS compared with the preoperative
assessment. Restenosis rates are elevated in the IJVs but very promising in the
AZY, suggesting the need to improve endovascular techniques in the former. The
results of this pilot study warrant a subsequent randomized control study. AIM: Chronic fatigue (CF) severely affects patients with multiple sclerosis
(MS), but its pathogenesis remains elusive and the effectiveness of available
treatments is modest. We aimed to evaluate the effect on CF of the balloon
dilatation of stenosing lesions affecting the main extracranial veins
configuring the chronic cerebrospinal venous insufficiency (CCSVI), a condition
strongly associated with MS.
METHODS: Thirty-one MS consecutive patients (16 males, age 46.2+/-9.4 years)
with associated CCSVI and CF underwent the endovascular procedure. Fatigue was
assessed using the Fatigue Severity Scale (FSS) and Modified Fatigue Impact
Scale (MFIS) at baseline (T0) and one (T1), six (T6) and twelve (T12) months
after the procedure. In ambulatory patients (N.=28), mobility was evaluated
using the 6-min walking test at T0 and T1.
RESULTS: and MFIS scores significantly improved from preoperative values, and
the positive trend was maintained at one year (FSS: T0=5.1+/-1.0 to
T12=3.5+/-1.8, P<0.001; MFIS-total score: T0=34.9+/-14.8 to T12=22.5+/-13.7,
P<0.001; MFIS-Physical subscale: T0=21.2+/-8.0 to T12=13.5+/-9.7 P<0.001;
MFIS-Cognitive subscale: T0=9.2+/-9.5 to T12=6.0+/-6.3, P=0.03;
MFIS-Psychosocial subscale: T0=4.5+/-2.1 to T12=2.5+/-2.1, P<0.001). Six-min
walking distance (6MWD) at T1 improved significantly (332+/-190m to 378+/-200m,
P=0.0002). In addition, an inverted correlation between 6MWD and MFIS-physical
subscale variations was found in the subgroup of patients (N.=8) with no lower
limb motor impairment (r=-0.74, P=0.035).
CONCLUSION: The reestablishment of cerebral venous return dramatically reduced
CF perception in a group of MS patients with associated CCSVI, suggesting that
CF is likely the symptom of CCSVI. The pyramidal pathway is frequently affected early on in multiple sclerosis (MS)
and impaired motor performance is a major cause of disability. Pyramidal tract
function can be assessed using transcranial magnetic stimulation (TMS). TMS
supports the diagnosis of MS, detecting corticospinal tract involvement and
monitoring its course with or without treatment. It has been never investigated
whether any relationship exists between the TMS outcome measure and minimally
invasive treatment of multiple severe extracranial stenosis, affecting the
principal ce rebrospinal venous segments in MS patients. We report the clinical
and transcranial magnetic stimulation follow-up of a patient during a relapse in
relapsing-remitting MS. She underwent percutaneous balloon angioplasty of the
associated chronic cerebrospinal venous insufficiency (CCSVI), due to membranous
obstruction of the proximal azygous vein, with severe stenosis of the left
internal jugular vein. Treatment of the associated CCSVI made a parallel
improvement in both clinical and neurophysiological parameters, allowing us to
avoid high dose steroid therapy. The relationship between the clinical and
neurophysiological course on the one hand, and haemodynamic correction of the
associated CCSVI on the other, calls for further exploration on a wider number
of patients. The impact of CCSVI on the different neuro-physiological parameters
has not been fully estimated, but the intriguing case here reported suggests
that it may be greater than previously assumed. The demonstration of a
modification of the cerebrovenous function with both clinical manifestation and
via TMS suggests that the hampered cerebral venous return may contribute to the
clinical course of MS. A chronic state of impaired venous drainage from the central nervous system,
termed chronic cerebrospinal venous insufficiency (CCSVI), is claimed to be a
pathologic phenomenon exclusively seen in multiple sclerosis (MS). This has
invigorated the causal debate of MS and generated immense interest in the
patient and scientific communities. A potential shift in the treatment paradigm
of MS involving endovascular balloon angioplasty or venous stent placement has
been proposed as well as conducted in small patient series. In some cases, it
may have resulted in serious injury. In this Point of View, we discuss the
recent investigations that led to the description of CCSVI as well as the
conceptual and technical shortcomings that challenge the potential relationship
of this phenomenon to MS. The need for conducting carefully designed and
rigorously controlled studies to investigate CCVSI has been recognized by the
scientific bodies engaged in MS research. Several scientific endeavors examining
the presence of CCSVI in MS are being undertaken. At present, invasive and
potentially dangerous endovascular procedures as therapy for patients with MS
should be discouraged until such studies have been completed, analyzed, and
debated in the scientific arena. From the earliest pathological studies the perivenular localization of the
demyelination in multiple sclerosis (MS) has been observed. It has recently been
suggested that obstructions to venous flow or inadequate venous valves in the
great veins in the neck, thorax and abdomen can cause damaging backflow into the
cerebral and spinal cord circulations. Paolo Zamboni and colleagues have
demonstrated abnormal venous circulation in some multiple sclerosis patients
using non-invasive sonography and invasive venography. Furthermore, they have
obtained apparent clinical improvement or stabilization by endovascular
ballooning of points of obstruction in the great veins in some, at least
temporarily. If non-invasive observations by others validate their initial
observations of a significantly increased prevalence of venous obstructions in
MS then trials of angioplasty/stenting would be justified in selected cases in
view of the biological plausibility of the concept. Chronic cerebrospinal venous insufficiency (CCSVI) is a syndrome characterized
by stenosies of the internal jugular and/or azygous veins (IJVs-AZ) with opening
of collaterals and insufficient drainage proved by reduced cerebral blood flow
and increased mean transit time in cerebral MRI perfusional study. The present
review is aimed to give a comprehensive overview of the actual status of the art
of the diagnosis and treatment of this condition. As far as the origin of venous
narrowing is concerned, phlebographic studies of the IJVs and AZ systems
demonstrated that venous stenoses were likely to be truncular venous
malformations; mostly, they are intraluminal defects such as malformed valve,
septa webs. CCSVI condition has been found to be strongly associated with
multiple sclerosis (MS), a disabling neurodegenerative and demyelinating disease
considered autoimmune in nature. In several epidemiological observations
performed at different latitudes on patients with different genetic backgrounds,
the prevalence of CCSVI in MS ranges from 56% to 100%. To the contrary, by using
venous MR and/or different Doppler protocols, CCSVI was not detected with the
same prevalence. Two pilot studies demonstrated the safety and feasibility in
Day Surgery of the endovascular treatment of CCSVI by means of balloon
angioplasty (PTA). It determines a significant reduction of postoperative venous
pressure. Restenosis rate was found out elevated in the IJVs, but negligible in
the AZ. However, PTA seems to positively influence clinical and QoL parameters
of the associated MS and warrants further randomized control trials. OBJECTIVES: The aim of this report is to assess the safety of endovascular
treatment for chronic cerebrospinal venous insufficiency (CCSVI). Although
balloon angioplasty and stenting seem to be safe procedures, there are currently
no data on the treatment of a large group of patients with this vascular
pathology.
METHODS: A total of 564 endovascular procedures (balloon angioplasty or, if this
procedure failed, stenting) were performed during 344 interventions in 331 CCSVI
patients with associated multiple sclerosis.
RESULTS: Balloon angioplasty alone was performed in 192 cases (55.8%), whereas
the stenting of at least one vein was required in the remaining 152 cases
(44.2%). There were no major complications (severe bleeding, venous thrombosis,
stent migration or injury to the nerves) related to the procedure, except for
thrombotic occlusion of the stent in two cases (1.2% of stenting procedures) and
surgical opening of femoral vein to remove angioplastic balloon in one case
(0.3% of procedures). Minor complications included occasional technical problems
(2.4% of procedures): difficulty removing the angioplastic balloon or problems
with proper placement of stent, and other medical events (2.1% of procedures):
local bleeding from the groin, minor gastrointestinal bleeding or cardiac
arrhythmia.
CONCLUSIONS: The procedures appeared to be safe and well tolerated by the
patients, regardless of the actual impact of the endovascular treatments for
venous pathology on the clinical course of multiple sclerosis, which warrants
long-term follow-up. PURPOSE: To evaluate the safety of endovascular treatment of chronic
cerebrovascular insufficiency (CCSVI) in patients with multiple sclerosis (MS).
METHODS: In a 1-year period, 461 MS patients (261 women; mean age 45.4 years,
range 21-79) with CCSVI underwent endovascular treatment of 1012 venous lesions
during 495 procedures [34 (6.9%) reinterventions]. While balloon angioplasty was
preferred, 98 stents were implanted in 76 patients for lesion recoil,
restenosis, or suboptimal dilation. The procedures were analyzed for incidences
of major adverse events (death, major bleeding, or clinical deterioration of
MS), access site complications, procedure-related complications, and procedural
safety-related variables (fluoroscopy and contrast times). The complication
rates were compared to published data for similar endovascular methods.
RESULTS: There were no deaths, major bleeding events, or clinical deterioration
of MS. Access site complications included limited groin hematoma (5, 1.0%);
there were no arteriovenous fistulas or puncture site infections. Systemic
complications included only rare cardiac arrhythmias (6, 1.2%).
Procedure-related complications included vein rupture (2, 0.4%), vein dissection
(15, 3.0%), acute in-stent/in-segment thrombosis (8, 1.6%), and acute recoil (1,
0.2%); there was no stent migration or fracture or distal embolization. Mean
fluoroscopy time was 22.7 minutes, and mean contrast volume was 136.3 mL.
CONCLUSION: Endovascular therapy appears to be a safe and reliable method for
treating CCSVI. Innovations such as purpose-specific materials and devices are
needed, as are case-controlled and randomized data to establish efficacy in
ameliorating MS symptoms. Chronic cerebrospinal venous insufficiency (CCSVI) is a hypothesis through which
cerebral venous drainage abnormalities contribute towards the pathogenesis of
multiple sclerosis. CCSVI venoplasty is already practised worldwide. We report
the case of a 33-year-old lady with multiple sclerosis who underwent left
internal jugular venoplasty resulting in iatrogenic jugular thrombosis requiring
open thrombectomy for symptom relief. This occurred without insertion of a stent
and while fully anticoagulated. Clinicians should be aware that endovenous
treatment of CCSVI could cause paradoxical deterioration of cerebral venous
drainage. Patients with complications post venoplasty are now presenting to
geographically distant vascular units. Recent reports have emerged suggesting that multiple sclerosis (MS) may be due
to abnormal venous outflow from the central nervous system, termed chronic
cerebrospinal venous insufficiency (CCSVI). These reports have generated strong
interest and controversy over the prospect of a treatable cause of this chronic
debilitating disease. This review aims to describe the proposed association
between CCSVI and MS, summarize the current data, and discuss the role of
endovascular therapy and the need for rigorous randomized clinical trials to
evaluate this association and treatment. OBJECTIVES: Chronic cerebrospinal venous insufficiency (CCSVI) is associated
with multiple sclerosis (MS). The objective of the study was to see if
percutaneous transluminal angioplasty (PTA) of duplex-detected lesions, of the
internal jugular and/or azygous veins, was safe, burdened by a significant
restenosis rate, and whether there was any evidence that treatment reduced MS
disease activity.
DESIGN: This was a case-control study.
MATERIALS: We studied 15 patients with relapsing-remitting MS and
duplex-detected CCSVI.
METHODS: Eight patients had PTA in addition to medical therapy (immediate
treatment group (ITG)), whereas seven had treatment with PTA after 6 months of
medical therapy alone (delayed treatment group (DTG)).
RESULTS: No adverse events occurred. At 1 year, there was a restenosis rate of
27%. Overall, PTA was followed by a significant improvement in functional score
compared with baseline (p < 0.02). The annualised relapse rate was 0.12% in the
ITG compared with 0.66% in the DTG (p = NS). Magnetic resoce imaging (MRI)
blindly demonstrates a trend for fewer T2 lesions in the ITG (p = 0.081),
corresponding to a 10% decrease in the ITG compared with a 23% increase in the
DTG over the first 6 months of the study.
CONCLUSIONS: This study further confirms the safety of PTA treatment in patients
with CCSVI associated with MS. The results, despite the significant rate of
restenosis, are encouraging and warrant a larger multicentre double-blinded,
randomised study. PURPOSE: To evaluate the safety of outpatient endovascular treatment in patients
with multiple sclerosis (MS) and chronic cerebrospinal venous insufficiency
(CCSVI).
MATERIALS AND METHODS: A retrospective analysis was performed to assess
complications occurring within 30 days of endovascular treatment of CCSVI. The
study population comprised 240 patients; 257 procedures were performed over 8
months. The indication for treatment in all patients was symptomatic MS. Of the
procedures, 49.0% (126 of 257) were performed in a hospital, and 51.0% (131 of
257) were performed in the office. Primary procedures accounted for 93.0% (239
of 257) of procedures, and repeat interventions accounted for 7% (18 of 257).
For patients treated primarily, 87% (208 of 239) had angioplasty, and 11% (26 of
239) had stent placement; 5 patients were not treated. Of patients with
restenosis, 50% (9 of 18) had angioplasty, and 50% (9 of 18) had stent
placement.
RESULTS: After the procedure, all but three patients were discharged within 3
hours. Headache after the procedure was reported in 8.2% (21 of 257) of
patients; headache persisted > 30 days in 1 patient. Neck pain was reported in
15.6% (40 of 257); 52.5% (21 of 40) of these patients underwent stent placement.
Three patients experienced venous thrombosis requiring retreatment within 30
days. Sustained intraprocedural arrhythmias were observed in three patients, and
two required hospital admission. One of these patients, who was being retreated
for stent thrombosis, was hospitalized because of a stress-induced
cardiomyopathy.
CONCLUSIONS: Endovascular treatment of CCSVI is a safe procedure; there is a
1.6% risk of major complications. Cardiac monitoring is essential to detect
intraprocedural arrhythmias. Ultrasonography after the procedure is recommended
to confirm venous patency and to identify patients experiencing acute venous
thrombosis. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central
nervous system (CNS) by an unknown pathogenesis. MR venography and postmortem
studies have demonstrated a topographic correspondence between multiple
sclerosis (MS) plaques and the cerebral venous system pathology. In recent
observational studies performed on patients from distinctive gene pools, the
prevalence of chronic cerebrospinal venous insufficiency (CCSVI) in MS ranged
from 56% to 100%. Endovascular treatment (percutaneous transluminal angioplasty
(PTA) with or without stenting) of CCSVI was reported to be feasible with a
minor complication rate. In 4 patients with different forms of multiple
sclerosis venography was performed that revealed stenosis of the proximal region
of the jugular vein (right or left). Percutaneous transluminal balloon
angioplasty (PTA) was performed in all patients. There were no complications and
mean stenosis was reduced after PTA from 59.75% to 36.75%. Follow-up included
clinical observations and magnetic resoce imaging (MRI). In all the cases we
observed positive remission of the disease, the first ever documented case of
MRI index improvement. PTA seems to be an effective treatment for patients with
CCVI and multiple sclerosis, However, randomized studies are warranted to
establish the efficacy of this new treatment for MS. Multiple sclerosis (MS) is a relapsing and progressive disorder of the central
nervous system. It is characterized most commonly by episodes of clinical
worsening, followed by clinical improvement. Pathologically, MS is associated
with focal areas of myelin destruction, inflammation, and axonal transection
("demyelinating plaques") in the brain and spinal cord. Traditionally, MS has
been considered an autoimmune disorder, with the primary pathophysiology arising
from an errant immune system. Recent work has raised the possibility that MS is
not caused primarily by an immune abnormality but may instead arise from venous
anomalies affecting the jugular and/or azygos venous systems. This condition has
been called chronic cerebrospinal venous insufficiency (CCSVI). It has been
proposed that CCSVI may be pathogenic in MS, causing venous back pressure and
iron deposition, with a secondary immune response. Some investigators have
proceeded to unblinded nonrandomized angioplasty and stenting procedures in
patients with CCSVI, with anecdotal reports of symptom improvement. Because of
conflicting data on the presence of CCSVI and the absence of controlled trials
of CCSVI intervention, the current standard of clinical care is neither to
evaluate multiple sclerosis (MS) patients for CCSVI anomalies, nor to intervene
with procedures to alter such anomalies. There is intense interest and ongoing
work to evaluate the presence of venous anomalies in MS patients as well as in
normal controls and patients with other neurologic conditions; to characterize
such anomalies, if present; and to further understand whether the concept of a
"backpressure" pathology is borne out by the evidence. If CCSVI is indeed a
pathogenic mechanism for some subset of the MS population, this would
dramatically change the focus of attention for therapeutic endeavors and
monitoring for this population and would bring MS therapeutics firmly into the
area of vascular intervention. On the other hand, the history of MS research
contains many novel and potentially paradigm-shifting ideas that were later
disproved by other investigators. Endovascular intervention for obstruction to venous drainage of the head and
neck is an established treatment for disorders such as superior vena cava
syndrome. Some patients with multiple sclerosis have been observed to have
anomalies of the veins draining the head and neck. It is possible that some
symptoms associated with multiple sclerosis may be secondary to disturbed venous
flow. In an uncontrolled clinical series of 40 patients who had been previously
diagnosed with multiple sclerosis, anomalies of the venous drainage of the head
and neck were observed, including venous stenoses of the internal jugular veins.
In 38 of 40 patients, venous stents were placed with restoration of luminal
dimensions and abrogation of the venous pressure gradient. The angiographic and
hemodynamic improvement was associated with improvement in symptomatology, most
particularly in cognitive and constitutional symptoms that may be related to
cerebrovenous flow. Serious complications included death in one subject and
stent embolization requiring open heart surgery in another. In conclusion, in
this series, endovascular intervention to correct venous stenosis associated
with multiple sclerosis was associated with improvement in symptoms possibly
related to disturbed venous hemodynamics. However, given the serious adverse
events in this small series, a randomized clinical trial is required to confirm
these findings, and to determine if the procedure has any effect on the
progression of multiple sclerosis, or untoward long-term adverse effects. A condition called "chronic cerebrospinal venous insufficiency" (CCSVI) has been
postulated to play a role in the pathogenesis of multiple sclerosis (MS). This
hypothesis implies that a complex pattern of extracranial venous stenosis
determines a venous reflux into the brain of MS patients, followed by increased
intravenous pressure, blood-brain barrier breakdown and iron deposition into the
brain parenchyma, thus triggering a local inflammatory response. In this review,
we critically analyze the scientific basis of CCSVI, the current literature on
the relationship between CCSVI and MS, as well as the ultrasound methodology
that has been claimed to provide evidence of impaired cerebral venous drainage.
We show that no piece of the CCSVI theory has a solid supportive scientific
evidence. The CCSVI appears to be a rather alien condition and its existence
should be definitely questioned. Finally, no proven (i.e., based on strict
scientific methodology and on the rules of evidence-based medicine) therapeutic
effect of the "liberation" procedure (unblocking the extracranial venous
obstruction using angioplasty) has been shown up to date. The debate regarding the possible link between chronic cerebrospinal venous
insufficiency and multiple sclerosis (MS) is continuously becoming more and more
contentious due to the current lack of level 1 evidence from randomized trials.
Regardless of this continued uncertainty surrounding the safety and efficacy of
this therapy, MS patients from Canada, and other jurisdictions, are traveling
abroad to receive central venous angioplasty and, unfortunately, some also
receive venous stents. They often return home with few instructions regarding
follow-up or medical therapy. In response we propose some interim, practical
recommendations for post-procedural surveillance and medical therapy, until
further information is available. An open study was conducted with the aim of reporting long-term clinical outcome
of endovascular treatment for chronic cerebrospinal venous insufficiency (CCSVI)
in patients with multiple sclerosis (MS). Twenty-nine patients with clinically
definite relapsing-remitting MS underwent percutaneous transluminal angioplasty
for CCSVI, outside a clinical relapse. All the patients were regularly observed
over at least two years before the first endovascular treatment and for at least
two years after it (mean post-procedure follow up 30.6±6.1 months). The
following clinical outcome measures were used: annual relapse rate and Expanded
Disability Status Scale (EDSS) score. All the patients were observed intensively
(mean 6 hours) on the day of the endovascular treatment to monitor for possible
complications (bleeding, shock, heart attack, death). We compared the annual
relapse rate before and after treatment (in the two years before and the two
years after the first endovascular treatment) and the EDSS score recorded two
years before versus two years after the treatment. Overall, 44 endovascular
procedures were performed in the 29 patients, without complications. Thirteen of
the 29 patients (45%) underwent more than one treatment session because of
venous re-stenosis: 11 and two patients underwent two and three endovascular
treatments respectively. The annual relapse rate of MS was significantly lower
post-procedure (0.45±0.62 vs 0.76±0.99; p=0.021), although it increased in four
patients. The EDSS score two years after treatment was significantly lower
compared to the EDSS score recorded at the examination two years before
treatment (1.98±0.92 vs 2.27±0.93; p=0.037), although it was higher in four
patients. Endovascular treatment of concurrent CCSVI seems to be safe and
repeatable and may reduce annual relapse rates and cumulative disability in
patients with relapsing-remitting MS. Randomized controlled studies are needed
to further assess the clinical effects of endovascular treatment of CCSVI in MS. PURPOSE: This study proposed to prospectively evaluate safety and clinical
changes in outpatient endovascular treatment in patients with multiple sclerosis
(MS) and chronic cerebrospinal venous insufficiency (CCSVI).
MATERIALS AND METHODS: Two hundred fifty-nine patients with MS were followed
with the Multiple Sclerosis Impact Scale (MSIS-29) before and for 1 and 6 months
after treatment of extracranial internal jugular vein and azygos vein stenoses
and occlusions using venous angioplasty, as well as stent placement in 2.5% of
patients. Before treatment, the patients were tested with magnetic resoce
(MR) venography and flow quantification.
RESULTS: We found statistically significant improvements in the MSIS-29 scores
(P < .01) at both 1 and 6 months. At 1 and 6 months, 67.9% and 53.6% were
improved on the physical scale, respectively, and 53.0% and 44.4% were improved
on the psychological scale, respectively. Women showed greater improvement than
did men on the physical scale at 6 months (P = .01). Patients with primary
progressive MS (PPMS) showed less improvement than did those with
relapsing-remitting MS (RRMS) on the psychological scale at 1 month, and
venoplasty treatment of more vein sites versus fewer vein sites showed greater
improvement on the physical scale at both 1 and 6 months. Fifteen patients
(6.3%) reported recurrent symptoms after clinical improvement and were treated
again. There was one serious adverse event, a deep venous thrombosis at the
catheter insertion site, which resolved with treatment.
CONCLUSIONS: Endovascular treatment of CCSVI in patients with MS appears to be a
safe procedure resulting in significant clinical improvement. OBJECTIVES: Condition known as chronic cerebrospinal venous insufficiency
(CCSVI) is characterized by insufficient cerebral vein drainage in patients with
multiple sclerosis (MS) and internal jugular vein (IJV), vertebral and/or azygos
veins stenoses. However, external compression on the IJV was not clearly
described as a potential cause of CCSVI. We aim to present a case of CCSVI in a
patient with MS caused by bilateral IJV inverted valves combined with IJV
external compression by carotid bulb.
METHODS: A 31-year-old female patient was admitted to our institute for IJV and
vertebral veins morphological and haemodynamical assessment after being treated
for MS for the last 14 years. Colour Doppler ultrasonography showed right IJV
prestenotic dilation and inverted valves in both IJV. Computerized tomography
angiography showed bilateral IJV compression by carotid bulb. Haemodynamical
Doppler parameters showed that external IJV compression significantly
contributed to CCSVI occurrence.
RESULTS: Bilateral IJV confluence percutaneous angioplasty (PTA) was done, and
the patient was discharged for further neurological examination. Partial carbon
dioxide pressure was significantly lower in the distal part of both IJV
following PTA and oxygen saturation increased.
CONCLUSION: In the case presented, PTA of the IJV confluence resulted in
haemodynamic improvement despite the presence of IJV external compression. OBJECTIVE: Chronic cerebrospinal venous insufficiency (CCSVI) was recently
described in patients with multiple sclerosis (MS). The hypothesis of the
vascular aetiology provides a new approach in the investigation and treatment of
MS.
METHODS: Our open-label study included 94 MS patients who fulfilled ultrasound
sonographic criteria required for CCSVI. The internal jugular and/or azygous
veins by a catheter venography were dilated.
RESULTS: In 34.8% of the patients unilateral, in 65.2% bilateral venous
abnormalities and in 2.1% no luminal obstructions were demonstrated. The patient
group with the higher disability score had a significantly higher number of
venous lesions (P < 0.005). Significant improvement of clinical disability in
relapsing-remitting patients was (P < 0.001) achieved. In our study no stents
were used. Re-stenosis occurred in 21.7% of the patients.
CONCLUSION: The number of venous narrowings is higher in more disabled patients.
A significant improvement in clinical disability in the relapsing-remitting
group was observed. BACKGROUND: Multiple sclerosis (MS) is a leading cause of neurological
disability in young adults. The most widely accepted hypothesis regarding its
pathogenesis is that it is an immune-mediated disease. It has been hypothesised
more recently that chronic venous congestion may be an important factor in the
pathogenesis of MS. This concept has been named 'chronic cerebrospinal venous
insufficiency' (CCSVI) and is characterised by stenoses of either the internal
jugular or azygos veins, or both. It is suggested that these stenoses restrict
the normal blood flow from the brain, causing the deposition of iron in the
brain and the eventual triggering of an auto-immune response. The proposed
treatment for CCSVI is percutaneous transluminal angioplasty, also known as the
'liberation procedure', which is claimed to improve the blood flow in the brain
thereby alleviating some of the symptoms of MS.
OBJECTIVES: To assess the effects of percutaneous transluminal angioplasty for
the treatment of CCSVI in people with MS.
SEARCH METHODS: We searched the following databases up to June 2012: The
Cochrane Multiple Sclerosis and Rare Diseases of the Central Nervous System
Group Specialised Register, CENTRAL in The Cochrane Library 2012, Issue 5,
MEDLINE (from 1946), EMBASE (from 1974), and reference lists of articles. We
also searched several online trials registries for ongoing trials.
SELECTION CRITERIA: Randomised controlled trials assessing the effects of
percutaneous transluminal angioplasty in adults with multiple sclerosis, that
have been diagnosed to have CCSVI.
DATA COLLECTION AND ANALYSIS: Our searches retrieved 159 references, six of
which were to ongoing trials. Based on assessment of the title or abstract, or
both, we excluded all of the studies, with the exception of one which was
evaluated following examination of the full text report. However, this study
also did not meet our inclusion criteria and was subsequently excluded.
MAIN RESULTS: No randomised controlled trials met our inclusion criteria.
AUTHORS' CONCLUSIONS: There is currently no high level evidence to support or
refute the efficacy or safety of percutaneous transluminal angioplasty for
treatment of CCSVI in people with MS. Clinical practice should be guided by
evidence supported by well-designed randomised controlled trials: closure of
some of the gaps in the evidence may be feasible at the time of completion of
the six ongoing clinical trials. We report female patient, age 51, with clinically definitive multiple sclerosis
(CDMS) since 1998, who underwent two PTA procedures with stent implantation for
CCSVI in 2010. Expanded disability status scale (EDSS) worsened since the
procedure from 4.5 to 6. Total number of three stents was implanted (two of them
in the right internal jugular vein). In six month time, in 2011, patient was
referred for independent examination by computer tomography (CT) phlebography
for right-sided neck pain. Dislocation of stents on the right side and
thrombosis of left sided stent was found. Conservative approach was used so far.
Our short report is showing possible complications of PTA and stenting in
jugular veins in so called CCSVI and bringing information about neurological
state (EDSS) worsening in a subject. Continuation of stent migration in the
future is probable, possibly resulting in pulmonary embolism with fatal risk for
the patient. We strongly ask for restriction of PTA procedure in so called
CCSVI, which concept was not proven to be relevant to MS. BACKGROUND: In 2009, Dr. Paolo Zamboni proposed chronic cerebrospinal venous
insufficiency (CCSVI) as a possible cause of multiple sclerosis (MS). Although
his theory and the associated treatment ("liberation therapy") received little
more than passing interest in the international scientific and medical
communities, his ideas became the source of tremendous public and political
tension in Canada. The story moved rapidly from mainstream media to social
networking sites. CCSVI and liberation therapy swiftly garnered support among
patients and triggered remarkable and relentless advocacy efforts. Policy makers
have responded in a variety of ways to the public's call for action.
DISCUSSION: We present three different perspectives on this evolving story, that
of a health journalist who played a key role in the media coverage of this
issue, that of a health law and policy scholar who has closely observed the
unfolding public policy developments across the country, and that of a medical
ethicist who sits on an expert panel convened by the MS Society of Canada and
the Canadian Institutes of Health Research to assess the evidence as it emerges.
SUMMARY: This story raises important questions about resource allocation and
priority setting in scientific research and science policy. The growing power of
social media represents a new level of citizen engagement and advocacy, and
emphasizes the importance of open debate about the basis on which such policy
choices are made. It also highlights the different ways evidence may be
understood, valued and utilized by various stakeholders and further emphasizes
calls to improve science communication so as to support balanced and informed
decision-making. RATIONALE: It is estimated that some hundreds of Canadian patients with multiple
sclerosis (MS) have journeyed abroad to avail themselves of 'liberation therapy'
(venoplasty) following the initial report by Zamboni et al in 2009. That study
also led to public pressure upon Departments of Health in Canadian Provinces to
fund the procedure. The present study was done in order to advise the Government
of Newfoundland and Labrador as to whether or not it should do so.
METHODS: We conducted an observational study of 30 MS subjects who had submitted
to venoplasty, using objective, semi-objective and subjective measures.
RESULTS: Significant subjective improvement was reported by half of the subjects
at three months, although the degree of perceived improvement was less at 12
months. The objective and semi-objective tests employed did not indicate
improvement in any area over the one-year follow-up period. Seven of the 29
subjects in whom CT venography was performed at the end of the study year were
found to have uni- or bilateral occlusion or >50% stenosis of at least one
cervical draining vein, but they showed no deterioration in their clinical
status compared to those in whom no venous occlusion nor stenosis was found.
CONCLUSION: No objective improvement was found at one year in thirty MS subjects
who had undergone venoplasty, although many reported a degree of subjective
benefit. BACKGROUND: It has been postulated that Multiple sclerosis (MS) stems from a
narrowing in the veins that drain blood from the brain, known medically as
chronic cerebrospinal venous insufficiency, or CCSVI. It has been proposed that
balloon angioplasty should alleviate the symptoms of MS. This procedure is also
known as The "Liberation Procedure". Accordingly, a clinical study was
undertaken to determine the effects of dexmedetomidine in patients undergoing
the liberation procedure.
AIMS: To assess the effectiveness of dexmedetomidine in providing adequate
sedation and pain relief for patients undergoing the liberation procedure.
SETTINGS AND DESIGN: A prospective, nonrandomized observational study of 60
consecutive adult patients undergoing the liberation procedure under monitored
anesthesia care (MAC) who will receive dexmedetomidine as an anesthetic agent.
METHODS: A total of 60 adult patients were enrolled in the study.
Dexmedetomidine was administered to all patients in a loading dose of 1 mcg/kg,
which was followed by a maintece dose of 0.2-0.5 mcg/kg/h. The evaluation of
quality of sedation was based on Ramsay Sedation and the quality of analgesia
was assessed using the visual analog scale. The following parameters were
measured continuously: heart rate, mean arterial pressure and hemoglobin oxygen
saturation. Patients were asked to answer the question, "How would you rate your
experience with the sedation you have received during surgery?" using a
seven-point Likert-like verbal rating scale.
STATISTICAL ANALYSIS: Repeated measurements were analyzed by repeated measures
ANOVA for HR and BP.
RESULTS: Most of our patients were satisfied with their sedation. In most of the
patients, MAP and HR dropped after the bolus dose of dexmedetomidine, and the
drop was statistically significant.
CONCLUSIONS: Dexmedetomidine can be used as a sole sedative agent in patients
undergoing the liberation procedure. PURPOSE: To investigate characteristics of cine phase contrast-calculated
cerebrospinal fluid (CSF) flow and velocity measures in patients with
relapsing-remitting (RR) multiple sclerosis (MS) receiving standard medical
treatment who had been diagnosed with chronic cerebrospinal venous insufficiency
(CCSVI) and underwent percutaneous transluminal angioplasty (PTA).
MATERIALS AND METHODS: This case-controlled, magnetic resoce (MR)
imaging-blinded study included 15 patients with RR MS who presented with
significant stenoses (≥50% lumen reduction on catheter venography) in the
azygous or internal jugular veins. Eight patients underwent PTA in addition to
medical therapy immediately following baseline assessments (case group) and
seven had delayed PTA after 6 months of medical therapy alone (control group).
CSF flow and velocity measures were quantified over 32 phases of the cardiac
cycle by a semiautomated method. Outcomes were compared between groups at
baseline and at 6 and 12 months of the study by mixed-effect model analysis.
RESULTS: At baseline, no significant differences in CSF flow or velocity
measures were detected between groups. At month 6, significant improvement in
flow (P<.001) and velocity (P = .013) outcomes were detected in the immediate
versus the delayed group, and persisted to month 12 (P = .001 and P = .021,
respectively). Within-group flow comparisons from baseline to follow-up showed a
significant increase in the immediate group (P = .033) but a decrease in the
delayed group (P = .024). Altered CSF flow and velocity measures were associated
with worsening of clinical and MR outcomes in the delayed group.
CONCLUSIONS: PTA in patients with MS with CCSVI increased CSF flow and decreased
CSF velocity, which are indicative of improved venous parenchyma drainage. Some multiple sclerosis (MS) patients reported an improvement after percutaneous
transluminal angioplasty (PTA) for chronic cerebrospinal venous insufficiency
(CCSVI), despite the lack of correspondence with objective outcome scores. The
objective was to assess neurologic and quality of life scores before and after
PTA for CCSVI in an observational study after a self-decided approach. 44
consecutive MS patients (21/23 M/F; median age 43 years, SD 9.8) who underwent
PTA were evaluated before endovascular treatment for CCSVI and after 12 months.
Neurologic outcome was assessed with EDSS, the annualized relapse rate (ARR) and
frequency of new lesions at MRI after PTA. Quality of life was evaluated through
the MSQoL-54 questionnaire. No modification in the ARR (p = 0.829), worsening of
disability status (p = 0.002) and new lesions at MRI in 29.6 % of patients were
found, in contrast to an improvement both in physical and mental domains of
MSQoL-54 (p = 0.003). Multiple logistic regression showed EDSS score before PTA
to be predictor of an increase of >10 points in MSQoL-54 mental domain (OR 0.52,
95 % CI, 0.31-0.89, p = 0.018). Spontaneously performed approach to CCSVI does
not improve clinical and MRI parameters, despite frequent subjective perception
of quality of life improvement. BACKGROUND: It has been recently hypothesised that chronic cerebrospinal venous
insufficiency (CCSVI) may be an important factor in the pathogenesis of multiple
sclerosis (MS). The proposed treatment for CCSVI is percutaneous transluminal
angioplasty, also known as the 'liberation procedure', which is claimed to
improve the blood flow in the brain, thereby alleviating some of the symptoms of
MS. Our objective was to determine the effects of percutaneous transluminal
angioplasty used for the treatment of CCSVI in people with MS.
METHODS: We searched the following databases up to June 2012 for randomised
controlled trials: The Cochrane Multiple Sclerosis and Rare Diseases of the
Central Nervous System Group Specialised Register, CENTRAL, in The Cochrane
Library 2012, Issue 5, MEDLINE (from 1946), EMBASE (from 1974) and reference
lists of articles. We also searched several online trials registries for ongoing
trials.
RESULTS: Our searches retrieved 159 references, six of which were related to
ongoing trials. No randomised controlled trials met our inclusion criteria.
CONCLUSIONS: There is currently no high level evidence to support or refute the
efficacy or safety of percutaneous transluminal angioplasty for treatment of
CCSVI in people with MS. Clinical practice should be guided by evidence
supported by well-designed randomised controlled trials: closure of some of the
gaps in the evidence may be feasible at completion of the six ongoing clinical
trials. Chronic cerebrospinal venous insufficiency (CCSVI) has been implicated as a
contributing factor to multiple sclerosis (MS). This theory is strongly debated
within the neurology and radiology communities. This report presents the case of
a 45-year-old man with known MS and suspected CCSVI who had undergone previous
internal jugular angioplasty and stenting. The patient reported dramatic
improvement of symptoms after intervention. The stent thrombosed despite
antithrombotic medication, and several endovascular interventions failed to
restore long-term patency. Open venous reconstruction of the internal jugular
vein was performed with a spiral graft from the saphenous vein. The patient's
symptoms improved for several weeks until the venous reconstruction occluded.
This case is the first reported open venous reconstruction for suspected CCSVI. OBJECTIVE: This study is the first in a series investigating the relationship
between autonomic nervous system dysfunction and chronic cerebrospinal venous
insufficiency in multiple sclerosis patients. We screened patients for the
combined presence of the narrowing of the internal jugular veins and symptoms of
autonomic nervous system dysfunction (fatigue, cognitive dysfunction, sleeping
disorders, headache, thermal intolerance, bowel/bladder dysfunction) and
determined systolic and diastolic blood pressure responses to balloon
angioplasty.
METHODS: The criteria for eligibility for balloon angioplasty intervention
included ≥ 50% narrowing in one or both internal jugular veins, as determined by
the magnetic resoce venography, and ≥ 3 clinical symptoms of autonomic
nervous system dysfunction. Blood pressure was measured at baseline and
post-balloon angioplasty.
RESULTS: Among patients who were screened, 91% were identified as having
internal jugular veins narrowing (with obstructing lesions) combined with the
presence of three or more symptoms of autonomic nervous system dysfunction.
Balloon angioplasty reduced the average systolic and diastolic blood pressure.
However, blood pressure categorization showed a biphasic response to balloon
angioplasty. The procedure increased blood pressure in multiple sclerosis
patients who presented with baseline blood pressure within lower limits of
normal ranges (systolic ≤ 105 mmHg, diastolic ≤ 70 mmHg) but decreased blood
pressure in patients with baseline blood pressure above normal ranges (systolic
≥ 130 mmHg, diastolic ≥ 80 mmHg). In addition, gender differences in baseline
blood pressure subcategories were observed.
DISCUSSION: The coexistence of internal jugular veins narrowing and symptoms of
autonomic nervous system dysfunction suggests that the two phenomena may be
related. Balloon angioplasty corrects blood pressure deviation in multiple
sclerosis patients undergoing internal jugular vein dilation. Further studies
should investigate the association between blood pressure deviation and internal
jugular veins narrowing, and whether blood pressure normalization affects
Patient's clinical outcomes. OBJECTIVE: We report the results of the investigation of safety and efficacy of
venous angioplasty in patients with multiple sclerosis (MS) with findings of
extracranial venous anomalies, considered hallmarks of chronic cerebrospinal
venous insufficiency (CCSVI), in a 2-phase study (ClinicalTrials.gov
NCT01450072).
METHODS: Phase 1 was an open-label safety study (10 patients); phase 2 was
sham-controlled, randomized, and double-blind (10 sham procedure, 9 treated).
All study patients fulfilled venous hemodynamic screening criteria indicative of
CCSVI. Assessment was at 1, 3, and 6 months postprocedure with MRI, clinical,
and hemodynamic outcomes. Primary endpoints were safety at 24 hours and 1 month,
venous outflow restoration >75% at 1 month, and effect of angioplasty on new
lesion activity and relapse rate over 6 months. Secondary endpoints included
changes in disability, brain volume, cognitive tests, and quality of life.
RESULTS: No perioperative complications were noted; however, one patient with
history of syncope was diagnosed with episodic bradycardia requiring placement
of a pacemaker before discharge. Doppler evidence-based venous hemodynamic
insufficiency severity score (VHISS) was reduced >75% compared to baseline in
phase 1 (at 1 month) but not phase 2. In phase 2, higher MRI activity
(cumulative number of new contrast-enhancing lesions [19 vs 3, p = 0.062] and
new T2 lesions [17 vs 3, p = 0.066]) and relapse activity (4 vs 1, p = 0.389)
were identified as nonsignificant trends in the treated vs sham arm over 6
months. Using analysis of covariance, significant cumulative new T2 lesions were
related to larger VHISS decrease (p = 0.028) and angioplasty (p = 0.01) over the
follow-up. No differences in other endpoints were detected.
CONCLUSION: Venous angioplasty is not an effective treatment for MS over the
short term and may exacerbate underlying disease activity.
CLASSIFICATION OF EVIDENCE: This is a Class I study demonstrating that clinical
and imaging outcomes are no better or worse in patients with MS identified with
venous outflow restriction who receive venous angioplasty compared to sham
controls who do not receive angioplasty. This study also includes a Class IV
phase 1 study of safety in 10 patients receiving the angioplasty procedure. |
Are the Fanconi anemia genes a part of the same signalling pathway? | The FA genes code for proteins that act in complexes to coordinate the repair of damaged DNA | Fanconi anaemia (FA) is an autosomal recessive inherited disorder associated
with a progressive aplastic anaemia, diverse congenital abnormalities and
cancer. The condition is genetically heterogeneous, with at least seven
complementation groups (A-G) described. Cells from individuals who are
homozygous for mutations in FA genes are characterized by chromosomal
instability and hypersensitivity to DNA interstrand crosslinking agents. These
features suggest a possible role for the encoded proteins in the recognition or
repair of these lesions, but neither their function nor whether they operate in
a concerted or discrete functional pathways is known. The recent cloning of the
FANCF and FANCE genes has allowed us to investigate the interaction of the
proteins encoded by five of the seven complementation groups of FA. We used the
yeast two-hybrid system and co-immunoprecipitation analysis to test the 10
possible pairs of proteins for direct interaction. In addition to the previously
described binding of FANCA to FANCG, we now demonstrate direct interaction of
FANCF with FANCG, of FANCC with FANCE and a weaker interaction of FANCE with
both FANCA and FANCG. These findings show that the newly identified FANCE
protein is an integral part of the FA pathway, and support the concept of a
functional link between all known proteins encoded by the genes that are mutated
in this disorder. These proteins may act either as a multimeric complex or by
sequential recruitment of subsets of the proteins in a common pathway that
protects the genomic integrity of mammalian cells. Fanconi anemia (FA) proteins function in a DNA damage response pathway that
appears to be part of the network including breast cancer susceptibility gene
products, BRCA1 and BRCA2. In response to DNA damage or replication signals, a
nuclear FA core complex of at least 6 FA proteins (FANCA, FANCC, FANCE, FANCF,
FANCG and FANCL) is activated and leads to monoubiquitination of the downstream
FA protein, FANCD2. One puzzling question for this pathway is the role of BRCA2.
A previous study has proposed that BRCA2 could be identical to two FA proteins:
FANCD1, which functions either downstream or in a parallel pathway; and FANCB,
which functions upstream of the FANCD2 monoubiquitination. Now, a new study
shows that the real FANCB protein is not BRCA2, but a previously uncharacterized
component of the FA core complex, FAAP95, suggesting that BRCA2 does not act
upstream of the FA pathway. Interestingly, the newly discovered FANCB gene is
X-linked and subject to X-inactivation. The presence of a single active copy of
FANCB and its essentiality for a functional FA-BRCA pathway make it a
potentially vulnerable component of the cellular machinery that maintains
genomic integrity. The Fanconi anemia (FA) gene family comprises at least 12 genes interacting in a
common pathway involved in DNA repair. To gain insight into the role of FA gene
inactivation occurring in tumors among the general population, we endogenously
targeted in cancer cells four FA genes that act at different stages of the FA
pathway. After successful mono-allelic deletion of all genes, the sequential
homozygous deletion was achieved only for FANCC and FANCG, acting upstream, but
not for BRCA2 or FANCD2, acting downstream in the FA pathway. Targeting of the
second allele in in BRCA2 and FANCD2 heterozygote clones resulted in redeletion
exclusively of the already defective allele in multiple instances (13x
concerning BRCA2, 25x concerning FANCD2), strongly suggesting a detrimental
phenotype. Unlike complete FANCD2 disruption, the mere reduction of FANCD2
protein levels had no discernible effect. In addition, we confirmed that human
cancer cells harboring the Seckel ATR mutation display impaired FANCD2
monoubiquitination and FANCD2 nuclear focus formation, as well as an increased
sensitivity to DNA interstrand-crosslinking agents. Nevertheless, these cells
were viable, indicating an ATR-independent function of FANCD2, distinct from its
major known functions, to be responsible for the detrimental effects of FANCD2
loss. In conclusion, we established the downstream FA genes FANCD2 and BRCA2 to
represent particularly vulnerable parts of the FA pathway, providing direct
evidence for the paradoxical assumption that their inactivation could be
predomitly selected against in cancer cells. This would explain why certain
FA gene defects, despite an apparent selection for FA pathway inactivation in
cancer, are rarely observed in tumors among the general population. Fanconi anemia (FA) is a rare human genetic disease caused by mutations in any
one of 13 known genes that encode proteins functioning in one common signaling
pathway, the FA pathway, or in unknown genes. One characteristic of FA is an
extremely high incidence of cancer, indicating the importance of the FA pathway
in tumor suppression. However, the role of this pathway in the development and
progression of human cancers in individuals who do not have FA has not been
clearly determined. Here, we report that elevated expression of what we believe
to be a novel splice variant of FA complementation group L (FANCL), which we
identified and named FAVL, can impair the FA pathway in non-FA human tumor cells
and act as a tumor promoting factor. FAVL expression was elevated in half of the
human carcinoma cell lines and carcinoma tissue samples tested. Expression of
FAVL resulted in decreased FANCL expression by sequestering FANCL to the
cytoplasm and enhancing its degradation. Importantly, this impairment of the FA
pathway by FAVL elevation provided human cancer cells with a growth advantage,
caused chromosomal instability in vitro, and promoted tumor development in a
xenograft mouse model. These data indicate that FAVL impairment of the FA
pathway likely contributes to the development of non-FA human cancers and
therefore add a challenging layer of complexity to the pathogenesis of human
cancer. We further believe that these data will prove useful for developing
additional tools for fighting human cancer. Fanconi anaemia (FA) is a rare, predomitly autosomal recessive syndrome (with
one X-linked form) that results in congenital defects, abnormal haematopoiesis
and a greatly increased risk of solid tumours in humans. Mutations in at least
14 different genes have been shown to cause FA, and several of these genes,
including FANCJ/BRIP1, FANCD1/BRCA2 and FANCN/PALB2, also predispose to breast
cancer in heterozygote carriers. The FA genes code for proteins that act in
complexes to coordinate the repair of damaged DNA, and thus the FA repair
network is intimately linked with hereditary breast cancer. Much remains to be
learnt about the functions and interactions of the FA proteins and one
experimental approach involves the generation of mice that are deficient in
various FA genes. Mouse models for FANCN/PALB2 have recently been generated,
including one reported in a recent issue of The Journal of Pathology. Given the
pivotal role of the PALB2 protein, which interacts with both BRCA1 and BRCA2,
these mice provide valuable insights into the FA phenotype and mechanisms of
tumourigenesis caused by disruption of the FA protein network. Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in
one of fifteen genes known to encode FA pathway components. In response to DNA
damage, nuclear FA proteins associate into high molecular weight complexes
through a cascade of post-translational modifications and physical interactions,
followed by the repair of damaged DNA. Hematopoietic cells are particularly
sensitive to the loss of these interactions, and bone marrow failure occurs
almost universally in FA patients. FA as a disease is further characterized by
cancer susceptibility, which highlights the importance of the FA pathway in
tumor suppression, and will be the focus of this review. Acute myeloid leukemia
is the most common cancer type, often subsequent to bone marrow failure.
However, FA patients are also at an extreme risk of squamous cell carcinoma
(SCC) of the head and neck and gynecological tract, with an even greater
incidence in those individuals who have received a bone marrow transplant and
recovered from hematopoietic disease. FA tumor suppression in hematopoietic
versus epithelial compartments could be mechanistically similar or distinct.
Definition of compartment specific FA activities is now critical to assess the
effects of today's bone marrow failure treatments on tomorrow's solid tumor
development. It is our hope that current therapies can then be optimized to
decrease the risk of maligt transformation in both hematopoietic and
epithelial cells. Here we review our current understanding of the mechanisms of
action of the Fanconi anemia pathway as it contributes to stress responses, DNA
repair and squamous cell carcinoma susceptibility. |
Are there clinical trials using stem cells for the treatment of cardiac disease? | Yes, there exists clinical trials for cardiac stem cell based treatment. | Atherosclerotic vascular disease becomes a clinical problem when there is
sufficient atherosclerotic plaque burden and/or endothelial dysfunction to cause
a limitation of nutrient blood flow to tissues. However, once myocardial
infarction has occurred, there is little, if any, way to stimulate the growth of
new blood vessels or cardiac muscle to replace that which has been lost. The
potential use of hematopoietic stem cells (HSCs) to treat cardiovascular disease
has recently been suggested from preclinical and clinical studies. HSCs are
precursors of all the blood cells, but they may also give rise to cells of the
vascular system, endothelial cells in the form of endothelial progenitor cells
(EPCs). Clinical trials have been conducted in patients with either acute
myocardial infarction or limb ischemia to determine the initial effectiveness
and safety of this treatment approach. These studies demonstrated the potential
clinical effectiveness of this stem cell approach to the treatment of patients
with acute myocardial ischemia and limb ischemia. Today, more preclinical
studies are planned to elucidate the mechanism by which transplanted stem cells
can home and differentiate into these endothelial cells and cardiac muscle
cells. At the same time, new clinical trials are planned to evaluate both
chronic, stable as well as acute myocardial ischemia and limb ischemia with
CD34+ and CD133+ stem cells, as well as with further selected EPCs and
mesenchymal stem cells. PURPOSE OF REVIEW: Stem cell therapy for treatment of cardiac disease has shown
therapeutic potential.
RECENT FINDINGS: A number of stem and progenitor populations have been
identified for potential use in cardiac repair. Each possesses a unique potency
that justifies consideration for use. Autologous, unfractionated bone marrow
cells or skeletal myoblasts were used in early clinical trails to evaluate
reparative effects on recent or record infarcts. In each case, evidence of
limited improvement in cardiac function was obtained. Myoblast grafts were
unexpectedly correlated with arrhythmias, thereby identifying a safety issue.
The small number of patients and the lack of randomized control groups preclude
conclusions regarding efficacy. Randomized controlled, intermediate-sized,
double-blind clinical trials must be undertaken to this end.
SUMMARY: Cellular therapy may be useful in the treatment of cardiac disease in
adults. Appropriate adaptations to meet unique requirements for treatment of
pediatric cardiovascular disease may be required. Bone marrow and skeletal
myoblasts do not promote true tissue regeneration in spite of observed
functional improvement. Trials using cells possessing true potential for
(trans)differentiation may elucidate the potential and value of this therapy as
a reparative modality. Development of optimal strategies for targeted delivery
consistent with pathobiology is of exception clinical relevance. Cardiomyopathy is a common clinical disorder affecting the heart muscle. This
disease process frequently leads to congestive heart failure and will often
progress to end-stage heart failure. Present standard of care treatment options
for cardiomyopathy include medical management, lifestyle changes, and surgical
procedures including left ventricular assist devices as a destiny therapy or
bridging to heart transplantation. Even despite advances in drug therapy,
mechanical assist devices, and organ transplantation, more than half of the
persons with cardiomyopathy will die within 5 years of diagnosis. Small
uncontrolled clinical trials have demonstrated cardiac stem cells as a treatment
option for cardiomyopathy. The theory for the individual or combined mechanism
of action for stem cells includes (1) transdifferentiation to blood vessels or
myocardium, (2) fusion with the native dysfunctional myocytes to augment
function, and (3) homing that may be a systemic or panacrine response for
recruiting other cells, and growth factors to help improve oxygen delivery and
myocardial function. The field of cardiac cell therapy is rapidly progressing to
gather more data with intermediate-size, double-blinded trials that will
demonstrate the safety and efficacy of cell therapy. Cell transplantation to repair or regenerate injured myocardium is a new
frontier in the treatment of cardiovascular disease. Most studies on stem cell
transplantation therapy in both experimental heart infarct and in phase-I human
clinical trials have focused on the use of undifferentiated stem cells. Based on
our previous observations demonstrating the presence of multipotent progenitor
cells in human adult skeletal muscle, in this study we investigated the capacity
of these progenitors to differentiate into cardiomyocytes. Here we show an
efficient protocol for the cardiomyogenic differentiation of human adult
skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid
directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro.
After Retinoic Acid treatment, cells expressed cardiomyocyte markers and
acquired spontaneous contraction. Functional assays exhibited cardiac-like
response to increased extracellular calcium. When cocultured with mouse
cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed
connexin43 and when transplanted into ischemic heart were detectable even 5
weeks after injection. Based on these results, we can conclude that human adult
skeletal muscle stem cells, if opportunely treated, can transdifferentiate into
cells of cardiac lineage and once injected into infarcted heart can integrate,
survive in cardiac tissue and improve the cardiac function. Ischemic heart disease is a major public health problem in the industrialized
and developing world. Despite advances in myocardial reperfusion strategies and
novel pharmacological approaches, ther apies directed towards the deleterious
consequences of acute and chronic myocardial ischemic damage remain limited. In
recent years the biological dogma of the heart as a "postmitotic organ" has been
challenged. Myocyte replication and myocardial regeneration have been documented
in the human heart after myocardial infarction and in chronic ischemic heart
failure. In addition, experimental animal studies and clinical trials suggest
that the transfer of stem and progenitor cells into the myocardium has a
favorable impact on tissue perfusion and contractile performance.
Neovascularization and myocyte formation have been described. Differentiation of
administered stem cells, cell fusion and release of paracrine signals by
injected stem cells are currently discussed as underlying mechanisms. Recently,
the mobilization of endogenous cardiac stem cells is debated as a potential
target of cardiac repair. This intriguing new knowledge generated by basic and
clinical scientists will lay the foundation for novel therapeutic strategies in
the near future and change clinical practice in cardiology. In this commentary,
we briefly review the characterization of the variety of stem cell populations
used for cardiac repair, discuss the potential mechanisms of cardiac
regenerative therapy, and evaluate the current clinical applications of this
innovative approach to treat ischemic heart disease. Heart failure is a major health problem on a global scale. Current therapies
include drug treatments, lifestyle modification, device therapy and heart
transplantation. However, the "holy grail" of heart failure treatment would be
to achieve widespread regeneration of diseased cardiac tissue. Examples of
regeneration of living tissue are present in nature and involve stem cells. The
two key defining properties of stem cells are their ability to renew themselves
through cell division and to differentiate into various cell types. Generally,
stem cells can be classified into embryonic or adult forms. Human adult stem
cells are ethically appealing and have already been used in clinical trials in a
variety of disease states. Bone marrow derived stem cells, skeletal myoblasts
and resident adult cardiac stem cells are being explored as potential cell types
for heart failure treatment. These cells can be delivered to the heart via a
number of routes. Several clinical trials using adult stem cell have shown
improvements in cardiac function, however, the mechanism of their action is
unclear and widespread tissue regeneration is not evident. A more comprehensive
understanding of regenerative physiology at the "benchside" combined with
ongoing investigations at the bedside, will be paramount in achieving the
ultimate goal of stem cell treatment-complete regeneration and repair of tissue. Cell therapy, particularly with stem cells, has created great interest as a
solution to the fact that there are limited treatments for postischemic heart
disease and none that can regenerate damaged heart cells to strengthen cardiac
performance. From the first efforts with myoblasts to recent clinical trials
with bone marrow-derived stem cells, early reports of cell therapy suggest
improvement in cardiac performance as well as other clinical end points. Based
on these exciting but tentative results, other stem cell types are being
explored for their particular advantages as a source of adult stem cells.
Autologous adipose-derived stem cells are multilinear and can be obtained
relatively easily in large quantities from patients; cardiac-derived stem cells
are highly appropriate for engraftment in their natural niche, the heart. Human
umbilical cord blood cells are potentially forever young and allogenic adult
mesenchymal stem cells appear not to evoke the graft versus host reaction. Human
embryonic stem cells are effective and can be scaled up for supply purposes. The
recent discovery of induced pluripotentcy in human adult stem cells, with only
three transcription factor genes, opens a whole new approach to making
autologous human pluripotent stem cells from skin or other available tissues.
Despite the excitement, stem cells may have to be genetically modified with heme
oxygenase, Akt or other genes to survive transplantation in a hypoxic
environment. Homing factors and hormones secreted from transplanted stem cells
may be more important than cells if they provide the necessary stimulus to
trigger cardiac regrowth to replace scar tissue. As we await results from larger
and more prolonged clinical trials, the science of stem cell therapy in cardiac
disease keeps progressing. Heart failure is a leading cause of death worldwide. Current therapies only
delay progression of the cardiac disease or replace the diseased heart with
cardiac transplantation. Stem cells represent a recently discovered novel
approach to the treatment of cardiac failure that may facilitate the replacement
of diseased cardiac tissue and subsequently lead to improved cardiac function
and cardiac regeneration. A stem cell is defined as a cell with the properties
of being clonogenic, self-renewing, and multipotent. In response to
intercellular signalling or environmental stimuli, stem cells differentiate into
cells derived from any of the three primary germ layers: ectoderm, endoderm, and
mesoderm, a powerful advantage for regenerative therapies. Meanwhile, a cardiac
progenitor cell is a multipotent cell that can differentiate into cells of any
of the cardiac lineages, including endothelial cells and cardiomyocytes. Stem
cells can be classified into three categories: (1) adult stem cells, (2)
embryonic stem cells, and (3) induced pluripotential cells. Adult stem cells
have been identified in numerous organs and tissues in adults, including
bone-marrow, skeletal muscle, adipose tissue, and, as was recently discovered,
the heart. Embryonic stem cells are derived from the inner cell mass of the
blastocyst stage of the developing embryo. Finally through transcriptional
reprogramming, somatic cells, such as fibroblasts, can be converted into induced
pluripotential cells that resemble embryonic stem cells. Four classes of stem
cells that may lead to cardiac regeneration are: (1) Embryonic stem cells, (2)
Bone Marrow derived stem cells, (3) Skeletal myoblasts, and (4) Cardiac stem
cells and cardiac progenitor cells. Embryonic stem cells are problematic because
of several reasons: (1) the formation of teratomas, (2) potential immunologic
cellular rejection, (3) low efficiency of their differentiation into
cardiomyocytes, typically 1% in culture, and (4) ethical and political issues.
As of now, bone marrow derived stem cells have not been proven to differentiate
reproducibly and reliably into cardiomyocytes. Skeletal myoblasts have created
in vivo myotubes but have not electrically integrated with the myocardium.
Cardiac stem cells and cardiac progenitor cells represent one of the most
promising types of cellular therapy for children with cardiac failure. Acute myocardial infarction (AMI) is one of the most significant causes of
morbidity and mortality worldwide. Stem cells represent an enormous chance to
rebuild damaged heart tissue. Correct definition of the cardiac progenitors is
necessary to understand heart development, and would pave the way for the use of
cardiac progenitors in the treatment of heart disease. Identifying, purifying
and differentiating native cardiac progenitor cells are indispensable if we are
to overcome congenital and adult cardiac diseases. To understand their
functions, physiology and action, cells are tested in animal models, and then in
clinical trials. But because clinical trials yield variable results, questions
about proper cardiac stem cells remain uswered. Transplanted stem cells
release soluble factors, acting in a paracrine fashion, which contributes to
cardiac regeneration. Cytokines and growth factors have cytoprotective and
neovascularizing functions, and may activate resident cardiac stem cells.
Understanding all these mechanisms is crucial to overcoming heart diseases. Cardiac stem cell therapy to promote engraftment of de novo beating cardiac
muscle cells in cardiomyopathies could potentially improve clinical outcomes for
many patients with congestive heart failure. Clinical trials carried out over
the last decade for cardiac regeneration have revealed inadequacy of current
approaches in cell therapy. Chief among them is the choice of stem cells to
achieve the desired outcomes. Initial enthusiasm of adult bone marrow stems
cells for myocyte regeneration has largely been relegated to paracrine-driven,
donor cell-independent, endogenous cardiac repair. However, true functional
restoration in heart failure is likely to require considerable myocyte
replacement. In order to match stem cell application to various clinical
scenarios, we review the necessity to preprime stem cells towards cardiac fate
before myocardial transplantation and if these differentiated stem cells could
confer added advantage over current choice of undifferentiated stem cells. We
explore differentiation ability of various stem cells to cardiac
progenitors/cardiomyocytes and compare their applicability in providing targeted
recovery in light of current clinical challenges of cell therapy. Stem cell therapy for cardiac disease is an exciting but highly controversial
research area. Strategies such as cell transplantation and reprogramming have
demonstrated both intriguing and sobering results. Yet as clinical trials
proceed, our incomplete understanding of stem cell behavior is made evident by
numerous unresolved matters, such as the mechanisms of cardiomyocyte turnover or
the optimal therapeutic strategies to achieve clinical efficacy. In this
Perspective, we consider how cardiac stem cell biology has led us into clinical
trials, and we suggest that achieving true cardiac regeneration in patients may
ultimately require resolution of critical controversies in experimental cardiac
regeneration. INTRODUCTION: Cardiovascular disease is a major cause of death worldwide.
Different medical and surgical therapeutic options are well established, but a
significant number of patients are not amenable to standard therapeutic options.
Cell-based therapies after clinical application have shown different results in
recent years. Here, we are giving a comprehensive overview on major available
clinical data regarding cell therapy.
BACKGROUND: Cell-based therapies and tissue engineering provide new promising
platforms to develop upcoming therapeutic options. Initial clinical trials were
able to generate promising results. A variety of different stem cell types have
been used for the clinical application. Different adult cardiac stem cells and
progenitor cells, including mesenchymal, CD34(+) and CD133(+) autologous human
bone marrow-derived stem cells (BMCs), human myoblasts, and peripheral
blood-derived stem and progenitor cells (PBSCs) have been used for the therapy
for end-stage heart failure. Future experiments will show the importance of
novel cell populations and clarify the mechanism causing cell therapy-mediated
observed effects.
CONCLUSION: Several clinical trials have reported on sole therapy, as well as
combined application of autologous adult stem cells with conventional
revascularization. The reported promising findings encourage further research in
the field of the translational research. Over the past 2 decades, there have been numerous stem cell studies focused on
cardiac diseases, ranging from proof-of-concept to phase 2 trials. This series
of papers focuses on the legacy of these studies and the outlook for future
treatment of cardiac diseases with stem cell therapies. The first section by
Drs. Rosen and Myerburg is an independent review that analyzes the basic science
and translational strategies supporting the rapid advance of stem cell
technology to the clinic, the philosophies behind them, trial designs, and means
for going forward that may impact favorably on progress. The second and third
sections were collected as responses to the initial section of this review. The
commentary by Drs. Francis and Cole discusses the review by Drs. Rosen and
Myerburg and details how trial outcomes can be affected by noise, poor trial
design (particularly the absence of blinding), and normal human tendencies
toward optimism and denial. The final, independent paper by Dr. Marbán takes a
different perspective concerning the potential for positive impact of stem cell
research applied to heart disease and future prospects for its clinical
application. (Compiled by the JACC editors). Therapy with mesenchymal stem cells is one of the promising tools to improve
outcomes after myocardial infarction. Adipose-derived stem cells (ASCs) are an
ideal source of mesenchymal stem cells due to their abundance and ease of
preparation. Studies in animal models of myocardial infarction have demonstrated
the ability of injected ASCs to engraft and differentiate into cardiomyocytes
and vasculature cells. ASCs secrete a wide array of angiogenic and
anti-apoptotic paracrine factors such as vascular endothelial growth factor,
hepatocyte growth factor, and insulin-like growth factor 1. ASCs are capable of
enhancing heart function, reducing myocardial infarction, promoting
vascularization, and reversing remodeling in the ischemically injured hearts.
Furthermore, several ongoing clinical trials using ASCs are producing promising
results for heart diseases. This article reviews the isolation, differentiation,
immunoregulatory properties, mechanisms of action, animal models, and ongoing
clinical trials of ASCs for cardiac disease. Despite substantial clinical advances over the past 65 years, cardiovascular
disease remains the leading cause of death in America. The past 15 years has
witnessed major basic and translational interest in the use of stem and
precursor cells as a therapeutic agent for chronically injured organs. Among the
cell types under investigation, adult mesenchymal stem cells are widely studied,
and in early stage, clinical studies show promise for repair and regeneration of
cardiac tissues. The ability of mesenchymal stem cells to differentiate into
mesoderm- and nonmesoderm-derived tissues, their immunomodulatory effects, their
availability, and their key role in maintaining and replenishing endogenous stem
cell niches have rendered them one of the most heavily investigated and
clinically tested type of stem cell. Accumulating data from preclinical and
early phase clinical trials document their safety when delivered as either
autologous or allogeneic forms in a range of cardiovascular diseases, but also
importantly define parameters of clinical efficacy that justify further
investigation in larger clinical trials. Here, we review the biology of
mesenchymal stem cells, their interaction with endogenous molecular and cellular
pathways, and their modulation of immune responses. Additionally, we discuss
factors that enhance their proliferative and regenerative ability and factors
that may hinder their effectiveness in the clinical setting. The prevalence of diabetes continues to increase world-wide and is a leading
cause of morbidity, mortality, and rapidly rising health care costs. Although
strict glucose control combined with good pharmacological and non-pharmacologic
interventions can increase diabetic patient life span, the frequency and
mortality of myocardial ischemia and infarction remain drastically increased in
diabetic patients. Therefore, more effective therapeutic approaches are urgently
needed. Over the past 15 years, cellular repair of the injured adult heart has
become the focus of a rapidly expanding broad spectrum of pre-clinical and
clinical research. Recent clinical trials have achieved favorable initial
endpoints with improvements in cardiac function and clinical symptoms following
cellular therapy. Due to the increased risk of cardiac disease, cardiac
regeneration may be one strategy to treat patients with diabetic cardiomyopathy
and/or myocardial infarction. However, pre-clinical studies suggest that the
diabetic myocardium may not be a favorable environment for the transplantation
and survival of stem cells due to altered kinetics in cellular homing, survival,
and in situ remodeling. Therefore, unique conditions in the diabetic myocardium
will require novel solutions in order to increase the efficiency of cellular
repair following ischemia and/or infarction. This review briefly summarizes some
of the recent advances in cardiac regeneration in non-diabetic conditions and
then provides an overview of some of the issues related to diabetes that must be
addressed in the coming years. Despite therapeutic advances, heart failure remains a common and serious event
characterized by initial and progressive loss of cardiac myocytes, a loss that
is currently untreatable. Cell therapy has emerged as a promising new approach
to the treatment of heart failure, with very encouraging experimental results.
Since 2000, when human stem cell therapy was first attempted in France, clinical
trials with adult stem cells (myoblasts, bone-marrow derived cells, mesenchymal
stem cells) have given variable results. The inconsistent and modest therapeutic
benefit observed in these studies is due more to paracrine effects than to the
hoped-for cell replacement, as adult stem cells do not turn into cardiomyocytes
and their survival rate after transplantation is very low. In order to be
effective, cell therapy should use heart muscle cells derived from pluri- or
multipotent cells (human embryonic stem cells, induced pluripotent stem cells,
resident cardiac cells), which are likely to have a higher survival rate in a
hostile biological environment and deteriorated tissue scaffold. Cardiac tissue
engineering assisted by otechnologies may eventually help to meet this
challenge. |
Is DNA methylation an epigenetic modification of chromatin related to gene expression? | Epigenetic changes such as DNA methylation alter gene expression at the level of transcription by upregulating, downregulating, or silencing genes completely. | Epigenetic changes are critical for development and progression of cancers,
including breast cancer. Significant progress has been made in the basic
understanding of how various epigenetic changes such as DNA methylation, histone
modification, miRNA expression, and higher order chromatin structure affect gene
expression. The present review will focus on methylation and demethylation of
histones. While the acetylation of histones has been at the forefront of
well-characterized post-translational modifications of histones, including the
development of inhibitors targeting de-acetylating enzymes, the past few years
have witnessed a dramatic increase in knowledge regarding the role of histone
methylation/demethylation. This is an exciting and rapidly evolving area of
research, with much promise for potential clinical intervention in several
cancers including breast cancer. We also summarize efforts to identity DNA
methylation signatures that could be prognostic and/or predictive markers in
breast cancer, focusing on recent studies using genome-wide approaches. Finally,
we briefly review the efforts made by both the National Institutes of Health
Epigenome Project and The Cancer Genome Atlas, especially highlighting the study
of breast cancer epigenetics, exciting technological advances, potential
roadblocks, and future directions. Besides known genetic aberrations, epigenetic alterations have emerged as common
hallmarks of many cancer types, including lung cancer. Epigenetics is a process
involved in gene regulation, mediated via DNA methylation, histone modification,
chromatin remodeling, and functional noncoding RNAs, which influences the
accessibility of the underlying DNA to transcriptional regulatory factors that
activate or repress expression. Studies have shown that epigenetic dysregulation
is associated with multiple steps during carcinogenesis. Since epigenetic
therapy is now in clinical use in hematopoietic diseases and undergoing trials
for lung cancer, a better understanding of epigenetic abnormalities is desired.
Recent technologies for high-throughput genome-wide analyses for epigenetic
modifications are promising and potent tools for understanding the global
dysregulation of cancer epigenetics. In this chapter, studies of epigenetic
abnormality and its clinical implication in lung cancers are discussed. Epigenetic modifications on the DNA sequence (DNA methylation) or on
chromatin-associated proteins (i.e., histones) comprise the "cellular
epigenome"; together these modifications play an important role in the
regulation of gene expression. Unlike the genome, the epigenome is highly
variable between cells and is dynamic and plastic in response to cellular stress
and environmental cues. The role of the epigenome, specifically, the methylome
has been increasingly highlighted and has been implicated in many cellular and
developmental processes such as embryonic reprogramming, cellular
differentiation, imprinting, X chromosome inactivation, genomic stability, and
complex diseases such as cancer. Over the past decade several methods have been
developed and applied to characterize DNA methylation at gene-specific loci
(using either traditional bisulfite sequencing or pyrosequencing) or its
genome-wide distribution (microarray analysis following methylated DNA
immunoprecipitation (MeDIP-chip), analysis by sequencing (MeDIP-seq), reduced
representation bisulfite sequencing (RRBS), or shotgun bisulfite sequencing).
This chapter reviews traditional bisulfite sequencing and shotgun bisulfite
sequencing approaches, with a greater emphasis on shotgun bisulfite sequencing
methods and data analysis. Epigenetic control, which includes DNA methylation and histone modifications,
leads to chromatin remodeling and regulated gene expression. Remodeling of
chromatin constitutes a critical interface of transducing signals, such as light
or nutrient availability, and how these are interpreted by the cell to generate
permissive or silenced states for transcription. CLOCK-BMAL1-mediated activation
of clock-controlled genes (CCGs) is coupled to circadian changes in histone
modification at their promoters. Several chromatin modifiers, such as the
deacetylases SIRT1 and HDAC3 or methyltransferase MLL1, have been shown to be
recruited to the promoters of the CCGs in a circadian manner. Interestingly, the
central element of the core clock machinery, the transcription factor CLOCK,
also possesses histone acetyltransferase activity. Rhythmic expression of the
CCGs is abolished in the absence of these chromatin modifiers. Here we will
discuss the evidence demonstrating that chromatin remodeling is at the
crossroads of circadian rhythms and regulation of metabolism and cellular
proliferation. DNA methylation patterns are characterized by highly conserved developmental
programs, but allow for divergent gene expression resulting from stochastic
epigenetic drift or divergent environments. Genome-wide methylation studies in
monozygotic (MZ) twins are providing insight into the extent of epigenetic
variation that occurs, irrespective of genotype. However, little is known about
the variability of DNA methylation patterns in adolescence, a period involving
significant and rapid physical, emotional, social, and neurodevelopmental
change. Here, we assessed genome-wide DNA methylation using the 450 K Illumina
BeadChip in a sample of 37 MZ twin pairs followed longitudinally since birth to
investigate: 1) the extent of variation in DNA methylation in identical genetic
backgrounds in adolescence and; 2) whether these variations are randomly
distributed or enriched in particular functional pathways. We also assessed
stability of DNA methylation over 3-6 months to distinguish stable trait-like
and variable state-like genes. A pathway analysis found high within-pair
variability in genes associated with development, cellular mechanisms, tissue
and cell morphology, and various disorders. Test-retest analyses performed in a
sub-sample of 8 twin pairs demonstrated enrichment in gene pathways involved in
organismal development, cellular growth and proliferation, cell signaling, and
particular disorders. The variability found in functional gene pathways may
plausibly underlie phenotypic differences in this adolescent MZ twin sample.
Furthermore, we assessed stability of methylation over 3-6 months and found that
some genes were stable while others were unstable, suggesting that the methylome
remains dynamic in adolescence and that dynamic sites tend to be organized in
certain gene pathways. Epigenetic changes such as DNA methylation and histone methylation and
acetylation alter gene expression at the level of transcription by upregulating,
downregulating, or silencing genes completely. Dysregulation of epigenetic
events can be pathological, leading to cardiovascular disease, neurological
disorders, metabolic disorders, and cancer development. Therefore, identifying
drugs that inhibit these epigenetic changes are of great clinical interest. In
this review, we summarize the epigenetic events associated with different
disorders and diseases including cardiovascular, neurological, and metabolic
disorders, and cancer. Knowledge of the specific epigenetic changes associated
with these types of diseases facilitates the development of specific inhibitors,
which can be used as epigenetic drugs. In this review, we discuss the major
classes of epigenetic drugs currently in use, such as DNA methylation inhibiting
drugs, bromodomain inhibitors, histone acetyl transferase inhibitors, histone
deacetylase inhibitors, protein methyltransferase inhibitors, and histone
methylation inhibitors and their role in reversing epigenetic changes and
treating disease. DNA methylation plays a critical role in the regulation of gene expression. Most
studies of DNA methylation have been performed in herbaceous plants, and little
is known about the methylation patterns in tree genomes. In the present study,
we generated a map of methylated cytosines at single base pair resolution for
Betula platyphylla (white birch) by bisulfite sequencing combined with
transcriptomics to analyze DNA methylation and its effects on gene expression.
We obtained a detailed view of the function of DNA methylation sequence
composition and distribution in the genome of B. platyphylla. There are 34,460
genes in the whole genome of birch, and 31,297 genes are methylated.
Conservatively, we estimated that 14.29% of genomic cytosines are
methylcytosines in birch. Among the methylation sites, the CHH context accounts
for 48.86%, and is the largest proportion. Combined transcriptome and
methylation analysis showed that the genes with moderate methylation levels had
higher expression levels than genes with high and low methylation. In addition,
methylated genes are highly enriched for the GO subcategories of binding
activities, catalytic activities, cellular processes, response to stimulus and
cell death, suggesting that methylation mediates these pathways in birch trees. |
Is Growth factor independence 1b (GFI1B) important for hematopoiesis? | Yes. Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Gfi-1b(-/-) embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b(-/-) embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. | Gfi-1 and Gfi-1b are novel proto-oncogenes identified by retroviral insertional
mutagenesis. By gene targeting, we establish that Gfi-1b is required for the
development of two related blood lineages, erythroid and megakaryocytic, in
mice. Gfi-1b(-/-) embryonic stem cells fail to contribute to red cells of adult
chimeras. Gfi-1b(-/-) embryos exhibit delayed maturation of primitive
erythrocytes and subsequently die with failure to produce definitive enucleated
erythrocytes. The fetal liver of mutant mice contains erythroid and
megakaryocytic precursors arrested in their development. Myelopoiesis is normal.
Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and
megakaryocyte development. In the search for genes expressed in hematopoietic stem cells, we identified
that the expression of Gfi-1B (growth factor independence-1B) is highly
restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1
and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc
finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid
maligcies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been
well characterized. In this study, we analyzed its function in human
hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic
progenitors induced a drastic expansion of erythroblasts in an
erythropoietin-independent manner. Expression of Gfi-1B did not promote
erythroid commitment, but enhanced proliferation of immature erythroblasts.
Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate
beyond proerythroblast stage and showed massive apoptosis. These biologic
effects of Gfi-1B were mediated through its zinc finger domain, but not by the
SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated
with sustained expression of GATA-2 but not of GATA-1, indicating a potential
link between Gfi-1B and GATA family regulators. Importantly, the function of
Gfi-1B to modulate transcription was dependent on promoter context. In addition,
activation of transcription of an artificial promoter was mediated through its
zinc finger domain. These findings establish Gfi-1B as a novel erythroid
regulator and reveal its specific involvement in the regulation of erythroid
cell growth through modulating erythroid-specific gene expression. Growth factor independence-1B (Gfi-1B) is a transcription factor with a highly
conserved transcriptional repressor snail-Gfi-1 (SNAG) domain and 6 zinc-finger
domains at the N- and C-terminus, respectively. Disruption of the Gfi-1B gene is
lethal in the embryo with failure to produce definitive enucleated erythrocytes.
In this study, we analyzed the role of Gfi-1B in human erythropoiesis. We
observed an increase of Gfi-1B expression during erythroid maturation of human
primary progenitor cells. We studied the consequences of variations in Gfi-1B
expression in 2 transformed cell lines (K562 and UT7 cells), as well as in
primary CD36(+)/GPA(-) progenitors. A knock-down of Gfi-1B delayed the terminal
differentiation of K562 and primary cells. Forced expression of Gfi-1B in UT7
and K562 cells led to an arrest of proliferation and an induction of erythroid
differentiation. Enforced expression of Gfi-1B in primary cells at the
colony-forming units-erythroid (CFU-E) stage led to a partial glycophorin A
(GPA) induction after erythropoietin (EPO) withdrawal but failed to protect
cells from apoptosis. Deletion of the SNAG repressor domain abolished
Gfi-1B-induced erythroid maturation, strongly suggesting that Gfi-1B acts in the
late stage of erythroid differentiation as a transcriptional repressor. Gfi-1B (growth factor independence-1B) gene is an erythroid-specific
transcription factor, whose expression plays an essential role in
erythropoiesis. Our laboratory has previously defined the human Gfi-1B promoter
region and shown that GATA-1 mediates erythroid-specific Gfi-1B transcription.
By further investigating the regulation of the Gfi-1B promoter, here we report
that (i) Gfi-1B transcription is negatively regulated by its own gene product,
(ii) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the
Gfi-1B promoter and (iii) Gfi-1B suppresses GATA-1-mediated stimulation of
Gfi-1B promoter through their protein interaction. These results not only
demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback
regulatory pathway in controlling the expression of the Gfi-1B gene, but also
provide the first evidence that Gfi-1B can exert its repression function by
acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site
of the target genes. Based on these data, we propose that this negative
auto-regulatory feedback loop is important in restricting the expression level
of Gfi-1B, thus optimizing its function in erythroid cells. Gfi1 is a transcriptional repressor essential for haematopoiesis and inner ear
development. It shares with its paralogue Gfi1b an amino-terminal SNAG repressor
domain and six carboxy-terminal zinc-finger motifs, but differs from Gfi1b in
sequences separating these domains. Here, we describe two knock-in mouse models,
in which the N-terminal SNAG repressor domain was mutated or in which the Gfi1
coding region was replaced by Gfi1b. Mouse mutants without an intact SNAG domain
show the full phenotype of Gfi1 null mice. However, Gfi1:Gfi1b knock-in mice
show almost normal pre-T-cell and neutrophil development, but lack properly
formed inner ear hair cells. Hence, our findings show that an intact SNAG domain
is essential for all functions of Gfi1 and that Gfi1b can replace Gfi1
functionally in haematopoiesis but, surprisingly, not in inner ear hair cell
development, demonstrating that Gfi1 and Gfi1b have equivalent and
domain-dependent, cell type-specific functions. Gfi1b and Gfi1 are 37- and 55-kDa transcriptional repressors that share common
features such as a 20-amino acid (aa) N-terminal SNAG domain, a nonconserved
intermediary domain, and 6 highly conserved C-terminal zinc fingers. Both gene
loci are under autoregulatory and cross-regulatory feedback control. We have
generated a reporter mouse strain by inserting the cDNA for green fluorescent
protein (GFP) into the Gfi1b gene locus which allowed us to follow Gfi1b
expression during hematopoiesis and lymphopoiesis by measuring green
fluorescence. We found highly dynamic expression patterns of Gfi1b in erythroid
cells, megakaryocytes, and their progenitor cells (MEPS) where Gfi1 is not
detected. Vice versa, Gfi1b could not be found in granulocytes, activated
macrophages, or their granulomonocytic precursors (GMPs) or in mature naive or
activated lymphocytes where Gfi1 is expressed, suggesting a complementary
regulation of both loci during hematopoiesis. However, Gfi1b was found to be
up-regulated in early stages of B-cell and in a subset of early T-cell
development, where Gfi1 is also present, suggesting that cross-regulation of
both loci exists but is cell-type specific. Gfi-1 and Gfi-1b are homologous transcriptional repressors involved in diverse
developmental contexts, including hematopoiesis and oncogenesis. Transcriptional
repression by Gfi proteins requires the conserved SNAG domain. To elucidate the
function of Gfi proteins, we purified Gfi-1b complexes and identified
interacting proteins. Prominent among these is the corepressor CoREST, the
histone demethylase LSD1, and HDACs 1 and 2. CoREST and LSD1 associate with
Gfi-1/1b via the SNAG repression domain. Gfi-1b further recruits these cofactors
to the majority of target gene promoters in vivo. Inhibition of CoREST and LSD1
perturbs differentiation of erythroid, megakaryocytic, and granulocytic cells as
well as primary erythroid progenitors. LSD1 depletion derepresses Gfi targets in
lineage-specific patterns, accompanied by enhanced histone 3 lysine 4
methylation at the respective promoters. Overall, we show that chromatin
regulatory proteins CoREST and LSD1 mediate transcriptional repression by Gfi
proteins. Lineage-restricted deployment of these cofactors through interaction
with Gfi proteins controls hematopoietic differentiation. To investigate the molecular effects of growth factor independence 1B (Gfi-1B),
a transcription factor essential for the development of hematopoietic cells and
differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B
overexpressing cell line K562 was cultured in the presence of Gfi-1B
target-specific small interfering RNA (siRNA). SiRNA treatment significantly
knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the
siRNA silencing protocol by colony-forming units ensured that it was not
cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down
Gfi-1B were analyzed by oligonucleotide microarray technology and based upon
rigorous statistical analysis of the data; relevant genes were chosen for
confirmation by reserve transcriptase-polymerase chain reaction, including
MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the
signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine
kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290,
JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in
Gfi-1B overexpressing cells compared to controls. Individual genes such as
ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing
cells. In addition, down-regulated transcripts showed cell signaling transcripts
for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30,
BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several
essential cell signalling factors, as well as transcriptional and
post-translational regulation genes were differentially expressed in cells that
overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our
data indicate that Gfi-1B signalling is important for commitment and maturation
of hematopoietic cell populations. Therapy-related myelodysplastic syndrome and acute myeloid leukemia
(t-MDS/t-AML) are late complications of cytotoxic therapy used in the treatment
of maligt diseases. The most common subtype of t-AML ( approximately 75% of
cases) develops after exposure to alkylating agents, and is characterized by
loss or deletion of chromosome 5 and/or 7 [-5/del(5q), -7/del(7q)], and a poor
outcome (median survival 8 months). In the University of Chicago's series of 386
patients with t-MDS/t-AML, 79 (20%) patients had abnormalities of chromosome 5,
95 (25%) patients had abnormalities of chromosome 7, and 85 (22%) patients had
abnormalities of both chromosomes 5 and 7. t-MDS/t-AML with a -5/del(5q) is
associated with a complex karyotype, characterized by trisomy 8, as well as loss
of 12p, 13q, 16q22, 17p (TP53 locus), chromosome 18, and 20q. In addition, this
subtype of t-AML is characterized by a unique expression profile (higher
expression of genes) involved in cell cycle control (CCNA2, CCNE2, CDC2),
checkpoints (BUB1), or growth (MYC), loss of expression of IRF8, and
overexpression of FHL2. Haploinsufficiency of the RPS14, EGR1, APC, NPM1, and
CTNNA1 genes on 5q has been implicated in the pathogenesis of MDS/AML. In
previous studies, we determined that Egr1 acts by haploinsufficiency and
cooperates with mutations induced by alkylating agents to induce myeloid
leukemias in the mouse. To identify mutations that cooperate with Egr1
haploinsufficiency, we used retroviral insertional mutagenesis. To date, we have
identified two common integration sites involving genes encoding transcription
factors that play a critical role in hematopoiesis (Evi1 and Gfi1b loci). Of
note is that the EVI1 transcription factor gene is deregulated in human AMLs,
particularly those with -7, and abnormalities of 3q. Identifying the genetic
pathways leading to t-AML will provide new insights into the underlying biology
of this disease, and may facilitate the identification of new therapeutic
targets. Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential
for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in
mice leads to embryonic lethality resulting from failure to produce definitive
erythrocytes, hindering the study of Gfi-1B function in adult hematopoiesis. We
here show that, in humans, Gfi-1B controls the development of erythrocytes and
megakaryocytes by regulating the proliferation and differentiation of bipotent
erythro-megakaryocytic progenitors. We further identify in this cell population
the type III transforming growth factor-beta receptor gene, TGFBR3, as a direct
target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth
factor-beta (TGF-beta) signaling as shown by the increase in Smad2
phosphorylation and its inability to associate to the transcription intermediary
factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to
specifically regulate erythroid differentiation, we propose that, by repressing
TGF-beta type III receptor (TbetaRIotaII) expression, Gfi-1B favors the
Smad2/TIF1-gamma interaction downstream of TGF-beta signaling, allowing immature
progenitors to differentiate toward the erythroid lineage. Growth factor-independence 1b (Gfi1b) is a zinc finger transcription factor
essential for erythroid and megakaryocytic development. To better understand
Gfi1b regulation and to know the implication of the level of expression of this
gene in human pathology, we have searched for promoter punctual sequence
variations in 214 patients with different hematological diseases. We found two
previously unknown congenital mutations at evolutionary conserved GATA and
octamer-binding (Oct) transcription factor sites. The Oct site mutation was also
found in five relatives of the patient. The GATA motif mutation reduced promoter
activity by 50% in vitro, while homozygous patients with the octamer site
mutation showed a four-to-five times increase of Gfi1b RNA in platelets.
Electrophoretic mobility shift analyses demonstrated that different protein
complexes bind to both sites and that binding is reduced by the mutations.
Finally, we found that GATA-1 and Oct-1 are the main components of each complex.
This study provides evidences of a new mechanism for Gfi1b repression. This is
also the first report of Gfi1b mutations with a functional implication; further
investigation and follow-up will clarify the involvement of these mutations in
hematological disease. Transcription factor Growth factor independence 1 (Gfi1) is required for
multilineage blood cell development, from stem and progenitor cells to
differentiated lymphoid and myeloid cells. Gfi1 expression is rapidly induced by
cytokines that control both the adaptive and innate immune systems. Gfi1 itself
represses the expression of genes implicated in cell survival, proliferation and
differentiation. Changes in Gfi1 expression and function have not only been
implicated in neutropenia, allergy, autoimmunity and hyperinflammatory
responses, but also in lymphoma and more recently in the development of
leukemia. In this study, we review how Gfi1 and its paralogue Gfi1b control the
development of blood cells, discuss how changes in Gfi1 and Gfi1b function
contribute to hematological disease and report on the molecular function of
these proteins. During development, haemogenesis occurs invariably at sites of vasculogenesis.
Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal
part of the dorsal aorta generate haematopoietic stem cells and are referred to
as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted
to this domain, and how the morphological transformation from endothelial to
haematopoietic is controlled are unknown. We show here that HoxA3, a gene
uniquely expressed in the embryonic but not yolk sac vasculature, restrains
haematopoietic differentiation of the earliest endothelial progenitors, and
induces reversion of the earliest haematopoietic progenitors into CD41-negative
endothelial cells. This reversible modulation of endothelial-haematopoietic
state is accomplished by targeting key haematopoietic transcription factors for
downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through
loss-of-function, and gain-of-function epistasis experiments, and the
identification of antipodally regulated targets, we show that among these
factors, Runx1 is uniquely able to erase the endothelial program set up by
HoxA3. These results suggest both why a frank endothelium does not precede
haematopoiesis in the yolk sac, and why haematopoietic stem cell generation
requires Runx1 expression only in endothelial cells. Lysine (K)-specific demethylase 1A (LSD1/KDM1A) has been identified as a
potential therapeutic target in solid cancers and more recently in acute myeloid
leukemia. However, the potential side effects of a LSD1-inhibitory therapy
remain elusive. Here, we show, with a newly established conditional in vivo
knockdown model, that LSD1 represents a central regulator of hematopoietic stem
and progenitor cells. LSD1 knockdown (LSD1-kd) expanded progenitor numbers by
enhancing their proliferative behavior. LSD1-kd led to an extensive expansion of
granulomonocytic, erythroid and megakaryocytic progenitors. In contrast,
terminal granulopoiesis, erythropoiesis and platelet production were severely
inhibited. The only exception was monopoiesis, which was promoted by LSD1
deficiency. Importantly, we showed that peripheral blood granulocytopenia,
monocytosis, anemia and thrombocytopenia were reversible after LSD1-kd
termination. Extramedullary splenic hematopoiesis contributed to the phenotypic
reversion, and progenitor populations remained expanded. LSD1-kd was associated
with the upregulation of key hematopoietic genes, including Gfi1b, Hoxa9 and
Meis1, which are known regulators of the HSC/progenitor compartment. We also
demonstrated that LSD1-kd abrogated Gfi1b-negative autoregulation by crossing
LSD1-kd with Gfi1b:GFP mice. Taken together, our findings distinguish LSD1 as a
critical regulator of hematopoiesis and point to severe, but reversible, side
effects of a LSD1-targeted therapy. Histone deacetylase inhibitors represent a family of targeted anticancer
compounds that are widely used against hematological maligcies. So far little
is known about their effects on normal myelopoiesis. Therefore, in order to
investigate the effect of histone deacetylase inhibitors on the myeloid
commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with
valproic acid (VPA). Our results demonstrate that VPA treatment induces H4
histone acetylation and hampers cell cycle progression in CD34(+) cells
sustaining high levels of CD34 protein expression. In addition, our data show
that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In
fact, we demonstrate that VPA treatment is able to induce the expression of
growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia
translocated to chromosome 3 protein (MLLT3), which are crucial regulators of
erythrocyte and megakaryocyte differentiation, and that the up-regulation of
these genes is mediated by the histone hyperacetylation at their promoter sites.
Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte
differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte
commitment only. As a whole, our data suggest that VPA sustains the expression
of stemness-related markers in hematopoietic stem/progenitor cells and is able
to interfere with hematopoietic lineage commitment by enhancing erythrocyte and
megakaryocyte differentiation and by inhibiting the granulocyte and
mono-macrophage maturation. Growth Factor Independence (Gfi) transcription factors play essential roles in
hematopoiesis, differentially activating and repressing transcriptional programs
required for hematopoietic stem/progenitor cell (HSPC) development and lineage
specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC),
myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC,
megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for
primitive hematopoiesis; however, little is known about the role of gfi1aa in
definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we
report the isolation and characterization of an additional hematopoietic gfi
factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and
definitive sites of hematopoiesis in zebrafish. Our functional analyses
demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive
and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences
markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b
silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for
definitive hematopoiesis. We determine the epistatic relationships between the
gfi factors and key hematopoietic transcription factors, demonstrating that
gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of
teleost HSPC. Our studies establish a comparative paradigm for the regulation of
hematopoietic lineages by gfi transcription factors. Growth factor independence 1b (GFI1B) is a DNA binding repressor of
transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at
midgestation very likely due to defects in erythro- and megakaryopoiesis. To
analyze the full functionality of Gfi1b, we used conditionally deficient mice
that harbor floxed Gfi1b alleles and inducible (Mx-Cre, Cre-ERT) or erythroid
specific (EpoR-Cre) Cre expressing transgenes. In contrast to the germline
knockout, EpoR-Cre mediated erythroid specific ablation of Gfi1b allows full
gestation, but causes perinatal lethality with very few mice surviving to
adulthood. Both the embryonic deletion of Gfi1b by EpoR-Cre and the deletion in
adult mice by Mx-Cre or Cre-ERT leads to reduced numbers of erythroid
precursors, perturbed and delayed erythroid maturation, anemia and
extramedullary erythropoiesis. Global expression analyses showed that the Hba-x,
Hbb-bh1 and Hbb-y embryonic globin genes were upregulated in Gfi1b deficient
TER119+ fetal liver cells over the gestation period from day 12.5-17.5 p.c. and
an increased level of Hbb-bh1 and Hbb-y embryonic globin gene expression was
even maintained in adult Gfi1b deficient mice. While the expression of Bcl11a, a
regulator of embryonic globin expression was not affected by Gfi1b deficiency,
the expression of Gata1 was reduced and the expression of Sox6, also involved in
globin switch, was almost entirely lost when Gfi1b was absent. These findings
establish Gfi1b as a regulator of embryonic globin expression and embryonic and
adult erythroid maturation. |
Has Revlimid been approved by the US Food and Drug Administration? | Yes, Revlimid has been approved by the US Food and Drug Administration for treatment of multiple myeloma. | The IMiDs represent a new proprietary class of thalidomide analogues that
possess greater potency and less toxicity than the parent compound. As a group,
these agents share the pharmacologic property of modulating cellular response to
ligand activation, the precise biologic effect of which is cell lineage and
stimulant-dependent. Lenalidomide (CC-5013; Revlimid), a second generation IMiD,
has shown significant erythropoietic activity in patients with lower risk MDS
that have failed or are not candidates for recombit erythropoietin treatment.
Unlike cytokine therapy, lenalidomide suppresses select MDS clones and enhances
erythropoietin receptor signaling to restore erythropoiesis. Activity is
greatest in patients with interstitial deletions involving chromosome 5q31.1. A
multicenter phase II study reported a 76 % overall transfusion response rate in
transfusion-dependent patients with deletion 5q, with 67 % achieving transfusion
independence after a median interval of 4.6 weeks of treatment. Cytogenetic
responses were observed in 73% of patients with complete cytogenetic remission
in 45% patients. Both transfusion response and cytogenetic response frequency
were independent of karyotype complexity, raising excitement that this new
treatment strategy might favorably alter the natural history of disease in
higher risk patients with deletion 5q. Lenalidomide was approved by the U.S.
Food and Drug Administration on December 27, 2005, for the treatment of IPSS Low
and intermediate-1 risk MDS patients with del(5q) abnormality. A phase III
Intergroup trial (ECOG 2905) will test the capacity to potentiate erythropoietin
response by comparing response to lenalidomide monotherapy to the combination of
darbepoetin and lenalidomide in non-deletion 5q MDS patients. Multiple Myeloma (MM) remains an incurable plasma cell maligcy in the bone
marrow (BM) despite conventional therapies as well as high-dose therapies with
stem cell support. Therefore novel biologically-based therapeutic approaches are
required. Recently, intensive laboratory and preclinical studies have identified
and validated therapeutic molecular targets in MM. In particular, recognition of
the biologic significance of the BM microenvironment in MM pathogenesis and as a
potential target for novel therapeutics has derived several promising
approaches. Novel FDA approved agents including thalidomide/thalomid, its
immunomodulatory derivatives lenalidomide/Revlimid, and proteasome inhibitor
bortezomib/Velcade are directed at molecular targets not only in MM cells but
also in its BM milieu, and have already achieved promising results in clinical
studies. Here we discuss the mechanisms of action of these novel drugs and their
clinical application, alone or combined with conventional or novel drugs. Lenalidomide, an IMiD drug (a novel type of immunomodulating drug) was recently
approved by the US Food and Drug Administration for the treatment of
transfusion-dependent anemia in patients with myelodysplastic syndromes (MDS)
and interstitial deletions of chromosome 5q [del(5q)]. This review examines the
clinical experience from the MDS-001 and MDS-003 clinical trials that led to
this approval, the results of biological correlates supporting the targets of
drug action, and the results from a non-del(5q) multicenter study (MDS-002).
Lenalidomide treatment resulted in both erythroid and cytogenetic responses in
the majority of patients with del(5q), accompanied by reductions in inflammatory
cytokine generation and marrow microvessel density and improvement in primitive
hematopoietic progenitor recovery. Central pathology review showed that
resolution of cytologic dysplasia was common in patients with del(5q) but was
infrequent in erythroid-responding patients without the chromosome 5 deletion.
These findings indicate that lenalidomide promotes erythropoiesis in lower-risk
MDS, with two apparently distinct mechanisms of action: suppression of the
ineffective del(5q) clone and promotion of effective erythropoiesis in
non-del(5q) MDS progenitors. These studies identified lenalidomide as a highly
active erythropoietic- and cytogenetic-remitting agent in lower-risk MDS
patients who otherwise would not be expected to benefit from recombit
erythropoietin therapy. The most common adverse reactions include dose-dependent
neutropenia and thrombocytopenia that are more pronounced in patients with
del(5q) in whom early suppression of the clone is expected. The bone marrow (BM) milieu confers drug resistance in multiple myeloma (MM)
cells to conventional therapies. Novel biologically based therapies are
therefore needed. Preclinical studies have identified and validated molecular
targeted therapeutics in MM. In particular, recognition of the biologic
significance of the BM microenvironment in MM pathogenesis and as a potential
target for novel therapeutics has already derived several promising approaches.
Thalidomide, lenalidomide (Revlimid), and bortezomib (Velcade) are directed not
only at MM cells but also at the BM milieu and have moved rapidly from the bench
to the bedside and United States Food and Drug Administration approval to treat
MM. PURPOSE: Lenalidomide (CC-5013, Revlimid; Celgene Corporation, Summit, NJ), a
thalidomide analogue, was granted approval by the U.S. Food and Drug
Administration (FDA) on June 29, 2006, for use in combination with dexamethasone
in patients with multiple myeloma (MM) who have received at least one prior
therapy. The FDA approved lenalidomide with a restricted distribution program,
RevAssist.
EXPERIMENTAL DESIGN: In two randomized, double-blind, multicenter studies, the
combination of lenalidomide and dexamethasone (LD) was compared with placebo and
dexamethasone (PD) in patients with MM who had received at least one prior
therapy. The primary endpoint was time to progression (TTP).
RESULTS: Following a prespecified interim analysis of TTP, an independent
data-monitoring committee advised the sponsor to halt the two studies. For both
studies, the interim analysis for efficacy revealed a statistically significant
longer TTP with LD than with PD. The most clinically relevant grade 3 and 4
adverse events that occurred more frequently in the LD arm were neutropenia,
thrombocytopenia, deep vein thrombosis, pulmonary embolism, and atrial
fibrillation. Thrombotic or thromboembolic events, including deep vein
thrombosis, pulmonary embolism, thrombosis, and intracranial venous sinus
thrombosis were reported more frequently in patients treated with LD than with
PD.
CONCLUSIONS: The FDA approved lenalidomide based on interim results from two
multicenter, placebo-controlled, randomized trials comparing the combination of
LD with PD that revealed a longer TTP with LD than with PD. The major toxicity
observed during these trials was myelosuppression. The serious toxicities
included thromboembolic events. Lenalidomide is only available under the
RevAssist Program. Lenalidomide was approved by the US Food and Drug Administration (FDA) for
treatment of transfusion-dependent lower-risk myelodysplastic syndrome patients
with deletion (del) (5q) alone or with additional karyotype abnormalities. The
approval was based on high rates of prolonged transfusion independence and
complete cytogenetic response in this subset. In lower-risk non-del(5q)
patients, meaningful erythroid responses also were reported with a low frequency
of cytogenetic improvement, although inferior to that observed in the del(5q)
patients. There is now a better understanding of the mechanism of the
karyotype-dependent drug action, explaining the disparate response rates and
frequency of myelosuppression. In del(5q) patients, lenalidomide suppresses the
clone by inhibiting the nuclear sequestration of the haplodeficient cell cycle
regulatory protein cdc25c, thereby promoting selective G2 arrest and apoptosis.
In non-del(5q) patients, lenalidomide enhances erythropoietin receptor
signaling. Future directions include use of biologic and molecular markers as
predictive tools to select patients and use of combination strategies to
overcome resistance to lenalidomide in del(5q) patients or enhance
erythropoiesis in non-del 5 patients. Prurigo nodularis is a chronic, relapsing neurodermatitis that is often
resistant to standard therapies with topical corticosteroids and oral
antihistamines. Thalidomide, while efficacious in treating recalcitrant cases of
prurigo nodularis, causes significant toxicity. Thalidomide-induced peripheral
neuropathy frequently results in drug discontinuation. Lenalidomide (Revlimid;
Celgene Corporation, Summit, NJ) is a derivative of thalidomide with less
neurotoxicity approved for the treatment of multiple myeloma and myelodysplastic
syndromes that has not been widely studied in dermatologic disorders. Here, we
report a case of refractory prurigo nodularis effectively treated with
lenalidomide. Given its favorable side-effect profile, lenalidomide may offer a
superior alternative to thalidomide in the treatment of this condition. In the past decade we have seen four new agents approved by the US Food and Drug
Administration for treatment of multiple myeloma: the proteasome inhibitor (PI)
bortezomib (Velcade), the immunomodulatory agents lenalidomide (Revlimid) and
thalidomide (Thalomid), and liposomal doxorubicin. These are commonly used in
the treatment of relapsed/refractory (R/R) multiple myeloma (MM), but there is
no universally accepted standard treatment. Salvage therapy must be tailored
according to an individual patient's clinical profile, with the risks and
potential effects of treatment-related adverse events being major determits
of the choice of therapy. Two novel agents in phase II studies to investigate
their potential for the treatment of R/R MM are carfilzomib, a selective,
irreversible next-generation PI, and pomalidomide, a next-generation thalidomide
analog. This review will discuss the side-effect profiles of the currently
approved immunomodulatory agents and bortezomib, as well as those of the newer
agents, carfilzomib and pomalidomide. In the past decade, immunomodulatory drugs have been approved by the US Food and
Drug Administration for the treatment of multiple myeloma (MM)-and a number of
emerging agents that target the cellular pathways or proteins involved in the
pathophysiology of MM are currently in development. Lenalidomide (Revlimid) and
pomalidomide induce apoptosis and sensitize MM cells while demonstrating
superior efficacy and better tolerability than thalidomide (Thalomid). Several
novel classes of drugs, including the histone deacetylase (HDAC) inhibitors,
heat shock protein (HSP) inhibitors, and monoclonal antibodies have been shown
to have activity in myeloma in early-stage clinical trials. HDAC inhibitors,
including vorinostat (Zolinza), panobinostat, and romidepsin (Istodax) are
thought to affect multiple pathways involved in MM and correct the deregulation
of genes involved in apoptosis and cell cycle arrest, thus potentially
sensitizing MM cells to apoptosis. HSP inhibitors (eg, tanespimycin) decrease MM
proliferation and suppress the long-term replicative potential of MM cells; they
may also sensitize MM cells to other anticancer agents. The humanized monoclonal
antibody elotuzumab induces antibody-dependent cell cytotoxicity-mediated
apoptosis. It is likely that in the near future the treatment armamentarium for
MM will undergo significant expansion as some of these additional target
pathways become validated. |
Which is the execution time (complexity) of the Smith-Waterman algorithm for the alignment of two sequences | The complexity of the Smith-Waterman dynamic programming algorithm is quadratic, that is, it runs in time proportional to the product of lengths of the sequences being aligned. | The sensitivity and selectivity of the FASTA and the Smith-Waterman protein
sequence comparison algorithms were evaluated using the superfamily
classification provided in the National Biomedical Research Foundation/Protein
Identification Resource (PIR) protein sequence database. Sequences from each of
the 34 superfamilies in the PIR database with 20 or more members were compared
against the protein sequence database. The similarity scores of the related and
unrelated sequences were determined using either the FASTA program or the
Smith-Waterman local similarity algorithm. These two sets of similarity scores
were used to evaluate the ability of the two comparison algorithms to identify
distantly related protein sequences. The FASTA program using the ktup = 2
sensitivity setting performed as well as the Smith-Waterman algorithm for 19 of
the 34 superfamilies. Increasing the sensitivity by setting ktup = 1 allowed
FASTA to perform as well as Smith-Waterman on an additional 7 superfamilies. The
rigorous Smith-Waterman method performed better than FASTA with ktup = 1 on 8
superfamilies, including the globins, immunoglobulin variable regions,
calmodulins, and plastocyanins. Several strategies for improving the sensitivity
of FASTA were examined. The greatest improvement in sensitivity was achieved by
optimizing a band around the best initial region found for every library
sequence. For every superfamily except the globins and immunoglobulin variable
regions, this strategy was as sensitive as a full Smith-Waterman. For some
sequences, additional sensitivity was achieved by including conserved but
nonidentical residues in the lookup table used to identify the initial region. The Smith-Waterman algorithm for local sequence alignment is one of the most
important techniques in computational molecular biology. This ingenious dynamic
programming approach was designed to reveal the highly conserved fragments by
discarding poorly conserved initial and terminal segments. However, the existing
notion of local similarity has a serious flaw: it does not discard poorly
conserved intermediate segments. The Smith-Waterman algorithm finds the local
alignment with maximal score but it is unable to find local alignment with
maximum degree of similarity (e.g. maximal percent of matches). Moreover, there
is still no efficient algorithm that answers the following natural question: do
two sequences share a (sufficiently long) fragment with more than 70% of
similarity? As a result, the local alignment sometimes produces a mosaic of
well-conserved fragments artificially connected by poorly-conserved or even
unrelated fragments. This may lead to problems in comparison of long genomic
sequences and comparative gene prediction as recently pointed out by Zhang et
al. (Bioinformatics, 15, 1012-1019, 1999). In this paper we propose a new
sequence comparison algorithm (normalized local alignment ) that reports the
regions with maximum degree of similarity. The algorithm is based on fractional
programming and its running time is O(n2log n). In practice, normalized local
alignment is only 3-5 times slower than the standard Smith-Waterman algorithm. Searching a database for a local alignment to a query under a typical scoring
scheme, such as PAM120 or BLOSUM62 with affine gap costs, is a computation that
has resisted algorithmic improvement due to its basis in dynamic programming and
the weak nature of the signals being searched for. In a query preprocessing
step, a set of tables can be built that permit one to (a) eliminate a large
fraction of the dynamic programming matrix from consideration and (b) to compute
several steps of the remainder with a single table lookup. While this result is
not an asymptotic improvement over the original Smith-Waterman algorithm, its
complexity is characterized in terms of some sparse features of the matrix and
it yields the fastest software implementation to date for such searches. The Smith-Waterman (SW) algorithm is a typical technique for local sequence
alignment in computational biology. However, the SW algorithm does not consider
the local behaviours of the amino acids, which may result in loss of some useful
information. Inspired by the success of Markov Edit Distance (MED) method, this
paper therefore proposes a novel Markov pairwise protein sequence alignment
(MPPSA) method that takes the local context dependencies into consideration. The
numerical results have shown its superiority to the SW for pairwise protein
sequence comparison. BACKGROUND: Due to its high sensitivity, the Smith-Waterman algorithm is widely
used for biological database searches. Unfortunately, the quadratic time
complexity of this algorithm makes it highly time-consuming. The exponential
growth of biological databases further deteriorates the situation. To accelerate
this algorithm, many efforts have been made to develop techniques in high
performance architectures, especially the recently emerging many-core
architectures and their associated programming models.
FINDINGS: This paper describes the latest release of the CUDASW++ software,
CUDASW++ 2.0, which makes new contributions to Smith-Waterman protein database
searches using compute unified device architecture (CUDA). A parallel
Smith-Waterman algorithm is proposed to further optimize the performance of
CUDASW++ 1.0 based on the single instruction, multiple thread (SIMT)
abstraction. For the first time, we have investigated a partitioned vectorized
Smith-Waterman algorithm using CUDA based on the virtualized single instruction,
multiple data (SIMD) abstraction. The optimized SIMT and the partitioned
vectorized algorithms were benchmarked, and remarkably, have similar performance
characteristics. CUDASW++ 2.0 achieves performance improvement over CUDASW++ 1.0
as much as 1.74 (1.72) times using the optimized SIMT algorithm and up to 1.77
(1.66) times using the partitioned vectorized algorithm, with a performance of
up to 17 (30) billion cells update per second (GCUPS) on a single-GPU GeForce
GTX 280 (dual-GPU GeForce GTX 295) graphics card.
CONCLUSIONS: CUDASW++ 2.0 is publicly available open-source software, written in
CUDA and C++ programming languages. It obtains significant performance
improvement over CUDASW++ 1.0 using either the optimized SIMT algorithm or the
partitioned vectorized algorithm for Smith-Waterman protein database searches by
fully exploiting the compute capability of commonly used CUDA-enabled low-cost
GPUs. The problem of finding an optimal structural alignment for a pair of
superimposed proteins is often amenable to the Smith-Waterman dynamic
programming algorithm, which runs in time proportional to the product of lengths
of the sequences being aligned. While the quadratic running time is acceptable
for computing a single alignment of two fixed protein structures, the time
complexity becomes a bottleneck when running the Smith-Waterman routine multiple
times in order to find a globally optimal superposition and alignment of the
input proteins. We present a subquadratic running time algorithm capable of
computing an alignment that optimizes one of the most widely used measures of
protein structure similarity, defined as the number of pairs of residues in two
proteins that can be superimposed under a predefined distance cutoff. The
algorithm presented in this article can be used to significantly improve the
speed-accuracy tradeoff in a number of popular protein structure alignment
methods. |
List mutations that are implicated in the Gray Platelet Syndrome. | GFI1B and NBEAL2 mutations are implicated in the Gray Platelet Syndrome. | Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is
characterized by large platelets that lack α-granules. Here we show that
mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain
protein, cause GPS and that megakaryocytes and platelets from individuals with
GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of
sucrose-gradient subcellular fractions of platelets indicated that NBEAL2
localizes to the dense tubular system (endoplasmic reticulum) in platelets. Next-generation RNA sequence analysis of platelets from an individual with
autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal
transcript reads, including intron retention, mapping to NBEAL2 (encoding
neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as
the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain
that is predicted to be involved in vesicular trafficking and may be critical
for the development of platelet α-granules. The gray platelet syndrome is a rare inherited bleeding disorder characterized
by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The
genetic defect responsible for gray platelet syndrome was recently identified in
biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with
inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All
of them underwent NBEAL2 DNA sequencing and evaluation of the platelet
phenotype, including a systematic assessment of the α-granule content by
immunofluorescence analysis for α-granule secretory proteins. We identified 9
novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included
missense, nonsense and frameshift mutations, as well as nucleotide substitutions
that altered the splicing mechanisms as determined at the RNA level. All the
individuals with NBEAL2 biallelic mutations showed almost complete absence of
platelet α-granules. Interestingly, the 13 individuals assumed to be
asymptomatic because carriers of a mutated allele had platelet macrocytosis and
significant reduction of the α-granule content. However, they were not
thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2
alterations, suggesting that other genetic defect(s) are responsible for their
platelet phenotype. Of note, these patients were characterized by a lower
severity of the α-granule deficiency than individuals with two NBEAL2 mutated
alleles. Our data extend the spectrum of mutations responsible for gray platelet
syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is
a genetic heterogeneous trait. In terms of practical applications, the screening
of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe
reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated
allele have mild laboratory abnormalities, suggesting that even
haploinsufficiency has an effect on platelet phenotype. Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with
macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to
loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here
a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit
splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and
their cargo, including von Willebrand factor (VWF), thrombospondin-1, and
platelet factor 4. The platelet α-granule membrane protein P-selectin is
expressed at 48% of wild-type levels and externalized upon platelet activation.
The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-)
platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later
stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown
by flow cytometry, platelet aggregometry, bleeding assays, and intravital
imaging of laser-induced arterial thrombus formation. Microscopic analysis
detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which
when cultured showed delayed maturation, decreased survival, decreased ploidy,
and developmental abnormalities, including abnormal extracellular distribution
of VWF. Our results confirm that α-granule secretion plays a significant role in
platelet function, and they also indicate that abnormal α-granule formation in
Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development,
and platelet production. The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding
disorder caused by a deficiency of alpha granules in platelets. We detected a
nonsense mutation in the gene encoding the transcription factor GFI1B (growth
factor independent 1B) that causes autosomal domit gray platelet syndrome.
Both gray platelets and megakaryocytes had abnormal marker expression. In
addition, the megakaryocytes had dysplastic features, and they were abnormally
distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant
GFI1B transcriptional activity in a domit-negative manner. Our studies show
that GFI1B, in addition to being causally related to the gray platelet syndrome,
is key to megakaryocyte and platelet development. |
Has single guide RNA been used on human cells? | Yes, sgRNA has been used in human cell lines. | The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to
genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
Here, we characterize SpCas9 targeting specificity in human cells to inform the
selection of target sites and avoid off-target effects. Our study evaluates >700
guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted
genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates
mismatches between guide RNA and target DNA at different positions in a
sequence-dependent manner, sensitive to the number, position and distribution of
mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA
methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize
off-target modification. To facilitate mammalian genome engineering
applications, we provide a web-based software tool to guide the selection and
validation of target sequences as well as off-target analyses. Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome
modifications across a range of eukaryotes. Here we engineer this system to
enable RNA-guided genome regulation in human cells by tethering transcriptional
activation domains either directly to a nuclease-null Cas9 protein or to an
aptamer-modified single guide RNA (sgRNA). Using this functionality we developed
a transcriptional activation-based assay to determine the landscape of
off-target binding of sgRNA:Cas9 complexes and compared it with the off-target
activity of transcription activator-like (TALs) effectors. Our results reveal
that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and
18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches,
respectively. By engineering a requirement for cooperativity through offset
nicking for genome editing or through multiple synergistic sgRNAs for robust
transcriptional activation, we suggest methods to mitigate off-target phenomena.
Our results expand the versatility of the sgRNA:Cas9 tool and highlight the
critical need to engineer improved specificity. Genome engineering in human pluripotent stem cells (hPSCs) holds great promise
for biomedical research and regenerative medicine. Recently, an RNA-guided,
DNA-cleaving interference pathway from bacteria [the type II clustered,
regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated
(Cas) pathway] has been adapted for use in eukaryotic cells, greatly
facilitating genome editing. Only two CRISPR-Cas systems (from Streptococcus
pyogenes and Streptococcus thermophilus), each with their own distinct targeting
requirements and limitations, have been developed for genome editing thus far.
Furthermore, limited information exists about homology-directed repair
(HDR)-mediated gene targeting using long donor DNA templates in hPSCs with these
systems. Here, using a distinct CRISPR-Cas system from Neisseria meningitidis,
we demonstrate efficient targeting of an endogenous gene in three hPSC lines
using HDR. The Cas9 RNA-guided endonuclease from N. meningitidis (NmCas9)
recognizes a 5'-NNNNGATT-3' protospacer adjacent motif (PAM) different from
those recognized by Cas9 proteins from S. pyogenes and S. thermophilus (SpCas9
and StCas9, respectively). Similar to SpCas9, NmCas9 is able to use a
single-guide RNA (sgRNA) to direct its activity. Because of its distinct
protospacer adjacent motif, the N. meningitidis CRISPR-Cas machinery increases
the sequence contexts amenable to RNA-directed genome editing. The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified
derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new
tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and
human cells. Here, we describe adaptations of these systems leading to
successful expression of the Cas9/sgRNA system in two dicot plant species,
Arabidopsis and tobacco, and two monocot crop species, rice and sorghum.
Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA
and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and
tobacco. The mutant GFP gene contained target sites in its 5' coding regions
that were successfully cleaved by a CAS9/sgRNA complex that, along with
error-prone DNA repair, resulted in creation of functional GFP genes. DNA
sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice
protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter
region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11,
were confirmed by DNA sequencing to contain mutated DNA sequences at the target
sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop
species bodes well for its near-term use as a facile and powerful means of plant
genetic engineering for scientific and agricultural applications. Sequence-specific control of gene expression on a genome-wide scale is an
important approach for understanding gene functions and for engineering genetic
regulatory systems. We have recently described an RNA-based method, CRISPR
interference (CRISPRi), for targeted silencing of transcription in bacteria and
human cells. The CRISPRi system is derived from the Streptococcus pyogenes
CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring
only the coexpression of a catalytically inactive Cas9 protein and a
customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA
elements complementary to the sgRNA and causes a steric block that halts
transcript elongation by RNA polymerase, resulting in the repression of the
target gene. Here we provide a protocol for the design, construction and
expression of customized sgRNAs for transcriptional repression of any gene of
interest. We also provide details for testing the repression activity of CRISPRi
using quantitative fluorescence assays and native elongating transcript
sequencing. CRISPRi provides a simplified approach for rapid gene repression
within 1-2 weeks. The method can also be adapted for high-throughput
interrogation of genome-wide gene functions and genetic interactions, thus
providing a complementary approach to RNA interference, which can be used in a
wider variety of organisms. The bacterial clustered regularly interspaced short palindromic repeats
(CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for
mammalian genetics, enabling the rapid generation of isogenic cell lines and
mice with modified alleles. Here, we describe a pooled, loss-of-function genetic
screening approach suitable for both positive and negative selection that uses a
genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression
cassettes were stably integrated into the genome, which enabled a complex mutant
pool to be tracked by massively parallel sequencing. We used a library
containing 73,000 sgRNAs to generate knockout collections and performed screens
in two human cell lines. A screen for resistance to the nucleotide analog
6-thioguanine identified all expected members of the DNA mismatch repair
pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide
identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A
negative selection screen for essential genes identified numerous gene sets
corresponding to fundamental processes. Last, we show that sgRNA efficiency is
associated with specific sequence motifs, enabling the prediction of more
effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a
powerful tool for systematic genetic analysis in mammalian cells. CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against
infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which
use small RNA guides (the crRNAs) to specify a cleavage site within the genome
of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving
activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using
synthetic single RNA guides, Cas9 can be reprogrammed to create specific
double-stranded DNA breaks in the genomes of a variety of organisms, ranging
from human cells to bacteria, and thus constitutes a powerful tool for genetic
engineering. Here we describe recent advancements in our understanding of type
II CRISPR-Cas immunity and how these studies led to revolutionary genome editing
applications. Engineered DNA-binding proteins that manipulate the human genome and
transcriptome have enabled rapid advances in biomedical research. In particular,
the RNA-guided CRISPR/Cas9 system has recently been engineered to create
site-specific double-strand breaks for genome editing or to direct targeted
transcriptional regulation. A unique capability of the CRISPR/Cas9 system is
multiplex genome engineering by delivering a single Cas9 enzyme and two or more
single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can
be used to simultaneously create multiple DNA breaks or to target multiple
transcriptional activators to a single promoter for synergistic enhancement of
gene induction. To address the need for uniform and sustained delivery of
multiplex CRISPR/Cas9-based genome engineering tools, we developed a single
lentiviral system to express a Cas9 variant, a reporter gene and up to four
sgRNAs from independent RNA polymerase III promoters that are incorporated into
the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently
expressed and can mediate multiplex gene editing and sustained transcriptional
activation in immortalized and primary human cells. This delivery system will be
significant to enabling the potential of CRISPR/Cas9-based multiplex genome
engineering in diverse cell types. CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to
protect bacteria from bacteriophage and other horizontally transmitted genetic
elements. Several CRISPR/Cas systems exist but the simplest variant, referred to
as Type II, has a single effector DNA endonuclease, called Cas9, which is guided
to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial
efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which
focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that
Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA
fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces
DNA cleavage leading to mutagenesis as a result of error prone non-homologous
end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high
throughput screening of genes in human cells for their relevance to a particular
phenotype and, more generally, for the targeted inactivation of specific genes,
in cell lines and in vivo in a number of model organisms. The latter aim seems
likely to be greatly enhanced by the recent development of Cas9 proteins from
bacterial species such as Neisseria meningitidis and Staphyloccus aureus that
are small enough to be expressed using adeno-associated (AAV)-based vectors that
can be readily prepared at very high titers. The evolving Cas9-based DNA editing
systems therefore appear likely to not only impact virology by allowing
researchers to screen for human genes that affect the replication of pathogenic
human viruses of all types but also to derive clonal human cell lines that lack
individual gene products that either facilitate or restrict viral replication.
Moreover, high titer AAV-based vectors offer the possibility of directly
targeting DNA viruses that infect discrete sites in the human body, such as
herpes simplex virus and hepatitis B virus, with the hope that the entire
population of viral DNA genomes might be destroyed. In conclusion, we believe
that the continued rapid evolution of CRISPR/Cas technology will soon have a
major, possibly revolutionary, impact on the field of virology. |
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