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How does Hst5 (histatin 5) affect infections by Candida glabrata? | Human salivary histatins, including histatin 5 (Hst 5), are small cationic proteins that are the major source of fungicidal activity of saliva | The incidence of disseminated candidiasis is increasing. Liver involvement is
frequent but rarely diagnosed. The authors report a case of disseminated
candidiasis due to Candida glabrata with liver metastases. The presence of
hepatic lesions was diagnosed by CT scan and parasitological examination of
liver abscess contents obtained by CT-scan-directed puncture-aspiration. The
outcome was favorable with amphotericin-B (cumulative dose of 1 g) and
flucytosin. Aspects of hepatic involvement in disseminated candidiasis is
discussed, together with the role of Candida glabrata in pathology of this type. Histatins are a group of small, cationic, antifungal peptides present in human
saliva. A previous molecular modeling analysis suggested structural similarity
between the Phe14-His15 and His18-His19 dipeptide sequences in histatin-5
(Hsn-5; a 24-amino-acid polypeptide) and the sequence of miconazole (one of the
azole-based antifungal therapeutic agents), implying that the mechanisms of
killing of Candida albicans by these two molecules may be similar. To further
elaborate on this observation, we have produced two variants of Hsn-5 in which
Phe14-His15 or His18-His19 dipeptide sequences were replaced by Ala-Ala
(F14A/H15A and H18A/H19A) to eliminate the phenyl and imidazole rings of the
side chains and assessed their candidacidal activities against C. albicans. In
addition, we tested azole-resistant C. albicans and Candida glabrata strains for
their susceptibilities to Hsn-5. Analysis of the purified recombit proteins
for their candidacidal activities indicated that both variants were
significantly less effective (the molar concentrations required to kill half of
the maximum number of cells [ED50s], approximately 67 and approximately 149
microM for F14A/H15A and H18A/H19A, respectively) than the unaltered Hsn-5
(ED50, approximately 8 microM) at killing C. albicans, suggesting that the two
dipeptide sequences are important for the candidacidal activity of Hsn-5.
Assessment of the candidacidal activity of Hsn-5 with the well-characterized
azole-resistant strains of C. albicans and C. glabrata, however, suggested that
the mode of action of histatins against Candida is distinct from that of
azole-based antifungal agents because Hsn-5 kills both azole-sensitive and
azole-resistant strains equally well. The present study shows that a number of basic antifungal peptides, including
human salivary histatin 5, a designed histatin analog designated dhvar4, and a
peptide from frog skin, PGLa, are active against amphotericin B-resistant
Candida albicans, Candida krusei, and Aspergillus fumigatus strains and against
a fluconazole-resistant Candida glabrata isolate. Fungicidal effects of histatin-5 against 26 oral isolates belonging to 5
non-albicans Candida species were examined. Fifty microM of histatin-5 killed
more than 95% of Candida tropicalis and Candida guilliermondii isolates and more
than 90% of Candida parapsilosis and Candida krusei. However, Candida glabrata
was less sensitive to the peptide (mean 62.9%). Our results, taken together,
demonstrated that histatin-5 possessed the fungicidal activity against Candida
species other than C. glabrata. Candida-associated denture stomatitis has a high rate of recurrence. Candida
biofilms formed on denture acrylic are more resistant to antifungals than
planktonic yeasts. Histatins, a family of basic peptides secreted by the major
salivary glands in humans, especially histatin 5, possess significant antifungal
properties. We examined antifungal activities of histatin 5 against planktonic
or biofilm Candida albicans and Candida glabrata. Candida biofilms were
developed on poly(methyl methacrylate) discs and treated with histatin 5
(0.01-100 microM) or fluconazole (1-200 microM). The metabolic activity of the
biofilms was measured by the XTT reduction assay. The fungicidal activity of
histatin 5 against planktonic Candida was tested by microdilution plate assay.
Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly
susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50%
reduction in the metabolic activity; biofilm) of 4.6 +/- 2.2, 6.9 +/- 3.7 and
1.7 +/- 1.5 microM, and IC(50) (planktonic cells) of 3.0 +/- 0.5, 2.6 +/- 0.1
and 4.8 +/- 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were
less susceptible to histatin 5, with 50% RMA of 31.2 +/- 4.8 and 62.5 +/- 0.7
microM, respectively. Planktonic C. glabrata was insensitive to histatin 5
(IC(50) > 100 microM). Biofilm-associated Candida was highly resistant to
fluconazole in the range 1-200 microM; e.g. at 100 microM only approximately 20%
inhibition was observed for C. albicans, and approximately 30% inhibition for C.
glabrata. These results indicate that histatin 5 exhibits antifungal activity
against biofilms of C. albicans and C. glabrata developed on denture acrylic. C.
glabrata is significantly less sensitive to histatin 5 than C. albicans. Candida albicans and Candida glabrata are predomit fungi associated with oral
candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with
high fungicidal activity against C. albicans, however many strains of C.
glabrata are resistant. Since Hst 5 requires fungal binding to cell wall
components prior to intracellular translocation, reduced Hst 5 binding to C.
glabrata may be the reason for its insensitivity. C. glabrata has higher surface
levels of β-1,3-glucans as compared with C. albicans; however these differences
did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly,
the biofilm matrix of C. glabrata contained significantly higher levels of
β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of
Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell
through polyamine transporters Dur3p and Dur31p that are uncharacterized in C.
glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher
killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm
matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate
limiting step is reduced uptake that can be overcome by expression of C.
albicans Dur proteins in C. glabrata. |
Which enzyme is deficient in Krabbe disease? | Galactocerebrosidase is an enzyme that is deficient in Krabbe disease (also known as globoid-cell leukodystrophy). This leads to accumulation of psychosine (galactosylsphingosine) primarily in oligodendrocytes. | Galactosylceramide beta-galactosidase cross reacting material was demonstrated
in brain, liver, and skin fibroblasts from patients with Krabbe disease. The
mutant enzyme was antigenically identical to the normal enzyme and exhibited
similar electrophoretic mobility. Normal quantities of the catalytically
deficient enzyme were measured in the patients' tissues by a sensitive single
radial immunodiffusion assay, indicating that the mutation is in structural gene
for the enzyme protein. 6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGal) has
been shown to be a specific fluorogenic substrate of galactocerebrosidase and to
facilitate the simple enzymatic diagnosis of Krabbe disease in human patients
and in twitcher mice. HMGal hydrolysis at pH 4.5 is optimally stimulated by
sodium taurocholate (0.25%) and oleic acid (0.05%) with a Km of 0.150, 0.04 and
0.03 mM, respectively for control mouse kidney, human fibroblasts and
leukocytes. In control samples, the specific activity (nmol/mg prot./h) for
HMGal is higher than for the natural substrate, galactocerebroside, and is
severely deficient in the twitcher mouse and in patients with Krabbe disease.
Comparative investigation of galactocerebrosidase activity in fibroblasts,
leukocytes and brain with radioactive and fluorogenic substrates reveals a good
agreement between the results of the two methods. Galactocerebroside (Gal-Cer)
is a competitive inhibitor of HMGal hydrolysis in mouse kidney homogenates while
GM1-ganglioside has no inhibitory effect in the same assay system. The
sensitivity and specificity of this fluorogenic substrate for
galactocerebrosidase provides a simple and rapid method for the diagnosis of
Krabbe disease, and for the purification of this enzyme from normal tissues. Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder
resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is
responsible for the lysosomal catabolism of galactosylceramide, a major lipid in
myelin, kidney and epithelial cells of small intestine and colon. We describe
the molecular cloning of human GALC cDNA and its expression in COS-1 cells.
Degenerate PCR primers, derived from N-terminal amino acid sequence from the 51
kDa band from human brain, were used to amplify cat testes RNA, and the
resulting product was used to screen human testes and brain libraries. Two
overlapping clones contained the total protein coding region, while additional
clones and PCR amplification were needed to obtain the complete 3' end of the
cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007
bp of open reading frame (coding for 669 amino acids), and 1741 bp of 3'
untranslated sequence. Modification of the sequence surrounding the initiation
codon to one more favorable for expression, resulted in a 6-fold increase in
GALC activity in transfected COS-1 cells. The isolation of this clone will
permit investigations into the causes for GALC deficiency in humans and
available animal models, development of more accurate tests for patient and
carrier identification, and evaluation of methods for effectively treating GALC
deficiency, initially using the animal models. Human galactocerebrosidase, the enzyme deficient in Krabbe disease, was
purified, through several hydrophobic column steps and gel filtration,
22,650-fold from human lymphocytes. Using information on its N-terminal and
internal amino acid sequences, and the polymerase chain reaction method, we
cloned a full-length cDNA for the enzyme. The deduced amino acid sequence
matched all amino acid sequences determined. The 3780 nucleotide sequence
included 2007 nucleotides which encoded a single chain peptide of 669 amino acid
residues with a 26 amino acid N-terminal signal peptide and six potential
asparagine-linked glycosylation sites. The galactocerebrosidase cDNA detected an
about 4 kb mRNA band material in human cultured skin fibroblasts. A nonsense
mutation was found at codon 369 (GAA-->TAA) in the coding sequence of cDNA
amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe
disease. Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a
series of hydrophobic affinity column chromatography steps. The activity was
enriched 176,000-fold from concentrated urine by only four columns, including
octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall
recovery was about 20% but only low amounts were obtained due to its low
abundance. The estimated final specific activities of several batches were
between 1 and 2 mmol/h per mg protein. The final purified fractions were
essentially free of other lysosomal enzyme activities. The most pure fractions
showed a series of bands between 50 and 53 kDa on sodium
dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have
identical N-terminal amino acid sequence. In addition, gel filtration of
partially purified GALC after disassociation showed one peak of activity
estimated to have a molecular mass near 50 kDa. GALC was also purified from
human brain and human placenta using the same methods demonstrating the
usefulness of this procedure in obtaining GALC from solid human tissues. In
addition to the bands migrating near 50 kDa from urine, there were also bands at
80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of
antipeptide antibodies, the 80 kDa band was demonstrated to have the same
N-terminal amino acids as the 50-53 kDa bands. The 30 kDa band had a unique
sequence. The relationship between the different molecular weight species
remains to be determined. The purification of GALC and the securing of amino
acid sequence information will aid in the cloning of the GALC gene. This enzyme
is deficient in human patients with Krabbe disease and several animal species. Krabbe disease is an autosomal recessive inherited demyelinating disease, which
is deficient in lysosomal enzyme, galactocerebrosidase. Pathophysiological
characteristics of this disease are extreme demyelination in white matter and
peripheral nerve, existence of globoid cells, absence of accumulation of main
substrates, i.e. galactocerebrosidase in tissues and accumulation of psychosine.
Molecular basis of this disease including isolation of a cDNA for human and
murine galactocerebrosidase and cloning of genome of this gene are reviewed. The
trial of gene therapy on twitcher, the mouse model of Krabbe disease, could
break through on therapy on this progressive demyelinating disease. Globoid-cell leukodystrophy (GLD) is an autosomal recessive inherited disorder
caused by the deficiency of galactocerebrosidase, the lysosomal enzyme
responsible for the degradation of the myelin glycolipid galactocerebroside.
Although the most common form of the disease is the classical infantile form
(Krabbe disease), later-onset forms also have been described. We have analyzed
the galactocerebrosidase gene in 17 patients (nine families) with late-onset GLD
and in 1 patient with classical Krabbe disease. Half of the patients were
heterozygous for the large gene deletion associated with the 502C-->T
polymorphism, the most common mutation in infantile patients. Several novel
mutations that result in deficient galactocerebrosidase activity were also
identified in these patients. They include the missense mutations R63H, G95S,
M101L, G268S, Y298C, and I234T; the nonsense mutation S7X; a one-base deletion
(805delG); a mutation that interferes with the splicing of intron 1; and a 34-nt
insertion in the RNA, caused by the aberrant splicing of intron 6. All of these
genetic defects are clustered in the first 10 exons of the galactocerebrosidase
gene and therefore affect the 50-kD subunit of the mature enzyme. Studies on the
distribution and enzymatic activity of the polymorphic alleles 1637T/C
(I546/T546) provided support for previous data that had indicated the existence
of two galactocerebrosidase forms with different catalytic activities in the
general population. Our data also indicate that the mutations occur
preferentially in the "low activity" 1637C allele. Galactocerebrosidase (GALC) is the lysosomal enzyme deficient in human and
certain animal species with globoid cell leukodystrophy (GLD) or Krabbe disease.
It catalyzes the hydrolysis of specific galactolipids including
galactosylceramide and psychosine. The GALC protein is found in very low amounts
in all tissues, which delayed its purification and the subsequent cloning of its
cDNA and gene. We previously published the exon-intron organization of the human
gene, but did not functionally analyze the 5' flanking region. We now provide a
description of this GC-rich region which includes one potential YY1 element and
one potential SP1 binding site. There are 13 GGC trinucleotides within the first
150 bp preceding the initiation codon. The 5' end of intron 1 contains six
potential Sp1 binding sites, one AP1 binding site, and eight AP2 binding sites.
A construct containing nucleotides -176 to -24 had the strongest promoter
activity using a vector containing the chloramphenicol acetyltransferase
reporter gene. We also provide evidence for the presence of inhibitory sequences
located immediately upstream of the promoter region, and within the first 234
nucleotides of intron 1. These elements together with a suboptimal nucleotide at
position +4 may explain the low level of GALC protein in all cell types. Globoid cell leukodystrophy (GCL or Krabbe disease) is a recessive disease
caused by mutations of the lysosomal enzyme galactocerebrosidase (GALC) and
twitcher is the murine model of GCL. We have prepared retroviral packaging cell
lines to transduce the GALC gene. Retroviral transduction restored GALC activity
in GCL fibroblasts and increased such activity to very high levels in
immortalized neural progenitor cells (ST14A cells). GALC activity was also
normalized in twitcher fibroblasts co-cultured with ST14A cells over-expressing
GALC, demonstrating that this enzyme is secreted and can be imported efficiently
by GALC-deficient cells. These results give the necessary background to evaluate
the therapeutic effect in twitcher of brain grafting of neural progenitor cells
engineered to release high levels of GALC. Galactocerebrosidase (GALC) is deficient in all tissues from human patients and
animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The
deficiency results in decreased lysosomal catabolism of certain galactolipids
including galactosylceramide and psychosine that are synthesized maximally
during myelination. According to current theories, the accumulation of
psychosine in humans and animals with GLD induces oligodendrocyte degeneration
and myelination ceases. Transduction of oligodendrocytes from twitcher mice with
a retroviral vector containing the GALC cDNA can correct the enzyme deficiency
in these cells. Our data show that twitcher astrocytes and oligodendrocytes can
internalize exogenous GALC, as well as donate the enzyme to the mutant glial
cells. Antibodies against human GALC localized the GALC antigen in retrovirally
transduced cells and cells receiving enzyme via cell to cell secretion and
uptake to the lysosomal fraction. In fact immunocytochemical studies in
transduced oligodendrocytes revealed that the GALC colocalizes in vesicles
lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells
with anti-GALC and a marker for oligodendrocytes demonstrated that, upon
differentiation, transduced, twitcher oligodendrocytes attained the normal
branched process configuration, while untransduced cells show only abnormal
morphology. Phenotype correction in mutant oligodendrocytes has also been
observed after enzyme transfer. These studies indicate that GALC activity
supplied to cultured oligodendrocytes from twitcher mice by different methods
can correct the pathological phenotype of these cells. BACKGROUND: Krabbe disease (globoid-cell leukodystrophy; GLD) is caused by
mutations in the GALC gene. Beta-galactocerebrosidase (GALC) is a specific
beta-galactosidase which is defective in GLD. About 90% of GLD patients have an
infantile course by fatal cerebral demyelination, but 10% have a later onset
(LOGLD) of symptoms and survive for one or several decades.
METHODS: Activities of GALC towards galactosylceramide (GC) and
galactosylsphingosine (psychosine; PS) were determined in white blood cells and
cultured fibroblasts derived from GLD patients and controls using
tritium-labelled natural substrates. In the galactosylsphingosine (psychosine)
beta-galactosidase (GALC-PS) assay, a thin layer chromatographic technique was
used to separate enzymatically released radioactive galactose.
RESULTS: Both galactosylceramide beta-galactosidase (GALC-GC) and GALC-PS
activities were reduced by at least 85% of the normal in all but 2 of the 10 GLD
patients studied. In particular, one 23-year-old severely demyelinated LOGLD
patient was strongly deficient (11% of the normal) in GALC-GC but apparently
normal for GALC-PS activity. This patient's GALC genotype was the
30-kb-deleted/502T allele combined with a wild-type allele in the 1637C
background known to slightly reduce GALC-GC activity. Further, of six LOGLD
patients, both of 62- and 63-year-old brothers had the deleted allele combined
with an 809G>A mutated 1637C allele. The sibs had strongly reduced GALC-GC and
GALC-PS activities but became clinically remarkable only in their 50s with a
severe mental downhill course in one of them.
CONCLUSIONS: A GALC genotype with one deleted and one polymorphic GALC
activity-reducing allele can lead to enzymatic and clinical signs of LOGLD in
the absence of marked GALC-PS deficiency. If an active PS hydrolysis in the
fibroblasts of a LOGLD patient also reflected such hydrolysis in the brain, the
psychosine hypothesis for GLD may need to be revised. Globoid cell leukodystrophy (Krabbe disease) is characterized by the
accumulation of a toxic metabolite, psychosine (galactosylsphingosine), which is
a substrate for the deficient enzyme (galactocerebroside beta-galactosidase).
This study underscores the possible role of psychosine in the effect of
inducible nitric oxide synthase (iNOS) -derived NO in the pathophysiology of
this demyelinating disease. For the first time, we provide evidence of the
expression of iNOS in CNS of Krabbe patient and show that the iNOS-expressing
cells in the CNS were astrocytes. Psychosine potentiated the LPS-induced
production of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in
primary rat astrocytes and regulated the cytokine-mediated production of NO in
C6 glioma and primary rat astrocyte. Psychosine induced cytokine-mediated
nuclear translocation of AP-1 and C/EBP by potentiating the expression of Fra-1
and C/EBP-delta proteins. This suggests that psychosine maintained or sustained
the cytokine-primed expression of iNOS by further potentiating the nuclear
translocation of AP-1 and C/EBP without modulating the cytokine-mediated
transcription activity of NF-kappaB. This study hypothesizes that accumulated
psychosine leads to production of cytokines and iNOS expression. The ensuing
excessive production of NO and ONOO- may play a role in pathogenesis of Krabbe
disease. This chapter describes in detail a practical procedure for the preparation of
radiolabeled galactocerebroside and its use in the assay of galactocerebrosidase
(GalCase), the enzyme deficient in globoid cell leukodystrophy (Krabbe disease).
The reference range for leukocytes and fibroblasts is 0.9-4.4 and 8-36 nmoles
substrate hydrolyzed per hour per milligram of protein, respectively. Because of
its low abundance this enzyme is difficult to assay in certain situations, such
as prenatal diagnosis by chorionic villus sampling. To obviate this a modified
assay is used where only the radiolabeled substrate is included in the
incubation. This provides a clear separation between affected samples and
unaffected controls. The methods detailed here should be reproducible in any
laboratory. Lipid rafts (LRs) are membrane realms characterized by high concentrations of
cholesterol and sphingolipids. Often, they are portrayed as scaffolds on which
many different signaling molecules can assemble their cascades. The idea of
rafts as scaffolds is garnering significant attention as the consequences of LR
disruption have been shown to be manifest in multiple signaling pathways. In
this study, LRs in the brain of the twitcher (TWI) mouse, a bona-fide model for
infant variants of human globoid cell leukodystrophy or Krabbe disease, were
investigated. This mouse has deficient activity of GALC
(beta-galactosylceramidase) that leads to a progressive accumulation of some
galactosyl-sphingolipids in the brain. We hypothesized that the accumulation of
psychosine (galactosyl-sphingosine) in the TWI CNS may result in the disruption
of rafts in different cell populations such as neurons and oligodendrocytes,
both cellular targets during disease. In this communication, we demonstrate that
psychosine specifically accumulates in LRs in the TWI brain and sciatic nerve
and in samples from brains of human Krabbe patients. It is also shown that this
accumulation is accompanied by an increase in cholesterol in these domains and
changes in the distribution of the LR markers flotillin-2 and caveolin-1.
Finally, we show evidence that this phenomenon may provide a mechanism by which
psychosine can exert its known inhibitory effect on protein kinase C. This study
provides a previously undescribed biophysical aspect for the mechanism of
pathogenesis in Krabbe disease. |
Are there Conserved Noncoding Elements (CNEs) in plant genomes? | The detailed view of conservation across angiosperms revealed not only high coding-sequence conservation but also a large set of previously uncharacterized intergenic conservation. Grass genes have dramatically fewer and much smaller CNSs than mammalian genes. Using an alignment-free information-retrieval approach, we have comprehensively identified all long identical multispecies elements (LIMEs), which include both syntenic and nonsyntenic regions, of at least 100 identical base pairs shared by at least two genomes. Using a comparative genomics approach with four dicotyledonous plant species (Arabidopsis thaliana, papaya [Carica papaya], poplar [Populus trichocarpa], and grape [Vitis vinifera]), we detected hundreds of CNSs upstream of Arabidopsis genes. We consequently compared the genomes of Arabidopsis thaliana and rice, which diverged about 200 million years ago, and identified 25 ultraconserved elements that are longer than 100 bp. Using a local sequence alignment set to deliver only significant alignments, we found one or more CNSs in the noncoding regions of the majority of genes studied. | Surveys for conserved noncoding sequences (CNS) among genes from monocot cereal
species were conducted to assess the general properties of CNS in grass genomes
and their correlation with known promoter regulatory elements. Initial
comparisons of 11 orthologous maize-rice gene pairs found that previously
defined regulatory motifs could be identified within short CNS but could not be
distinguished reliably from random sequence matches. Among the different
phylogenetic footprinting algorithms tested, the VISTA tool yielded the most
informative alignments of noncoding sequence. VISTA was used to survey for CNS
among all publicly available genomic sequences from maize, rice, wheat, barley,
and sorghum, representing >300 gene comparisons. Comparisons of orthologous
maize-rice and maize-sorghum gene pairs identified 20 bp as a minimal length
criterion for a significant CNS among grass genes, with few such CNS found to be
conserved across rice, maize, sorghum, and barley. The frequency and length of
cereal CNS as well as nucleotide substitution rates within CNS were consistent
with the known phylogenetic distances among the species compared. The
implications of these findings for the evolution of cereal gene promoter
sequences and the utility of using the nearly completed rice genome sequence to
predict candidate regulatory elements in other cereal genes by phylogenetic
footprinting are discussed. Ultraconserved elements, sequences with 100% identity with no insertions or
deletions between genomes, have been found in both vertebrate and invertebrate
genomes; whether plant genomes contain ultraconserved elements, however, is
unknown. We consequently compared the genomes of Arabidopsis thaliana and rice,
which diverged about 200 million years ago, and identified 25 ultraconserved
elements that are longer than 100 bp. Similar to those previously found,
ultraconserved elements in plants tend to occur in clusters and locate at
noncoding regions; nevertheless, they have many distinct features. For instance,
the longest ultraconserved element between the 2 plant genomes is 1491 bp, much
longer than the longest one (779 bp) between the human and rodent genomes. Some
biological implications are discussed, but the functions of these plant
ultraconserved elements and the reasons why they are practically frozen during
the evolution of millions of years remain a mystery. Ultraconserved elements (UCEs) are DNA sequences that are 100% identical (no
base substitutions, insertions, or deletions) and located in syntenic positions
in at least two genomes. Although hundreds of UCEs have been found in animal
genomes, little is known about the incidence of ultraconservation in plant
genomes. Using an alignment-free information-retrieval approach, we have
comprehensively identified all long identical multispecies elements (LIMEs),
which include both syntenic and nonsyntenic regions, of at least 100 identical
base pairs shared by at least two genomes. Among six animal genomes, we found
the previously known syntenic UCEs as well as previously undescribed nonsyntenic
elements. In contrast, among six plant genomes, we only found nonsyntenic LIMEs.
LIMEs can also be classified as either simple (repetitive) or complex
(nonrepetitive), they may occur in multiple copies in a genome, and they are
often spread across multiple chromosomes. Although complex LIMEs were found in
both animal and plant genomes, they differed significantly in their composition
and copy number. Further analyses of plant LIMEs revealed their functional
diversity, encompassing elements found near rRNA and enzyme-coding genes, as
well as those found in transposons and noncoding DNA. We conclude that despite
the common presence of LIMEs in both animal and plant lineages, the evolutionary
processes involved in the creation and maintece of these elements differ in
the two groups and are likely attributable to several mechanisms, including
transfer of genetic material from organellar to nuclear genomes, de novo
sequence manufacturing, and purifying selection. Conserved noncoding sequences (CNSs) in DNA are reliable pointers to regulatory
elements controlling gene expression. Using a comparative genomics approach with
four dicotyledonous plant species (Arabidopsis thaliana, papaya [Carica papaya],
poplar [Populus trichocarpa], and grape [Vitis vinifera]), we detected hundreds
of CNSs upstream of Arabidopsis genes. Distinct positioning, length, and
enrichment for transcription factor binding sites suggest these CNSs play a
functional role in transcriptional regulation. The enrichment of transcription
factors within the set of genes associated with CNS is consistent with the
hypothesis that together they form part of a conserved transcriptional network
whose function is to regulate other transcription factors and control
development. We identified a set of promoters where regulatory mechanisms are
likely to be shared between the model organism Arabidopsis and other dicots,
providing areas of focus for further research. Here, we describe the construction of a phylogenetically deep, whole-genome
alignment of 20 flowering plants, along with an analysis of plant genome
conservation. Each included angiosperm genome was aligned to a reference genome,
Arabidopsis thaliana, using the LASTZ/MULTIZ paradigm and tools from the
University of California-Santa Cruz Genome Browser source code. In addition to
the multiple alignment, we created a local genome browser displaying multiple
tracks of newly generated genome annotation, as well as annotation sourced from
published data of other research groups. An investigation into A. thaliana gene
features present in the aligned A. lyrata genome revealed better conservation of
start codons, stop codons, and splice sites within our alignments (51% of
features from A. thaliana conserved without interruption in A. lyrata) when
compared with previous publicly available plant pairwise alignments (34% of
features conserved). The detailed view of conservation across angiosperms
revealed not only high coding-sequence conservation but also a large set of
previously uncharacterized intergenic conservation. From this, we annotated the
collection of conserved features, revealing dozens of putative noncoding RNAs,
including some with recorded small RNA expression. Comparing conservation
between kingdoms revealed a faster decay of vertebrate genome features when
compared with angiosperm genomes. Finally, conserved sequences were searched for
folding RNA features, including but not limited to noncoding RNA (ncRNA) genes.
Among these, we highlight a double hairpin in the 5'-untranslated region
(5'-UTR) of the PRIN2 gene and a putative ncRNA with homology targeting the LAF3
protein. |
Which is the localization of the RIFIN family of proteins? | Plasmodium falciparum rifin proteins are mainly surface-expressed. Data has shown that while A-type RIFINs were found to be associated with the parasite and transported to the surface of infected erythrocytes via Maurer's clefts, B-type RIFINs appeared to be mostly retained inside the parasite. | Plasmodium falciparum rifin proteins, belonging to the largest known family of
variable infected-erythrocyte surface-expressed proteins encoded by rif genes,
were recently shown to be capable of inducing a strong immune response in P.
falciparum-infected adults living in an area in Gabon where malaria is endemic.
In the present study, the levels of antirifin antibodies were analyzed in serum
obtained from 60 children from the same area who were admitted to hospital and
diagnosed with severe malaria. High antirifin antibody concentrations in these
individuals correlated significantly with their capacity to rapidly clear their
parasites from the circulation after the start of chemotherapy. A doubling of
antirifin antibody concentrations reduced the clearance time by 5 h (95%
confidence interval, 4.1 to 6.9 h). In the same group of children, who were
followed up for 2 years, antirifin antibody levels did not correlate with a
reduced rate of reinfection or with a delay in the time to the first
reinfection. However, the initial antirifin antibody levels were sustained over
the study period. The likelihood that these antibodies could confer a certain
degree of protection against malaria is supported by our findings of
statistically higher levels of antirifin antibodies to all four rifin proteins
in a group of 42 asymptomatic parasitemic children. Proteins on the surface of parasite-infected erythrocytes (PIESPs) have been one
of the major focuses of malaria research due to their role in pathogenesis and
their potential as targets for immunity and drug intervention. Despite intense
scrutiny, only a few surface proteins have been identified and characterized. We
report the identification of two novel surface proteins from Plasmodium
falciparum-infected erythrocytes. Surface proteins were fractionated through
biotin-streptavidin interaction and analyzed by shotgun proteomics. From a list
of 36 candidates, two were selected for further characterization. The surface
location of both proteins was confirmed by confocal microscopy using specific
antibodies. PIESP1 and PIESP2 are unlikely to be associated with knobs, the
protrusions on the parasite-infected erythrocyte (PIE) surface. In contrast to
other known PIESPs, such as PfEMP1 and Rifin, these novel proteins are encoded
by single copy genes, highly conserved across Plasmodium ssp., making them good
targets for interventions with a broad specificity to various P. falciparum
isolates. The surfaces of the infected erythrocyte (IE) and the merozoite, two
developmental stages of malaria parasites, expose antigenic determits to the
host immune system. We report on surface-associated interspersed genes (surf
genes), which encode a novel polymorphic protein family, SURFINs, present on
both IEs and merozoites. A SURFIN expressed in 3D7 parasites, SURFIN4.2, was
identified by mass spectrometric analysis of peptides cleaved off the surface of
live IEs with trypsin. SURFINs are encoded by a family of 10 surf genes,
including three predicted pseudogenes, located within or close to the
subtelomeres of five of the chromosomes. SURFINs show structural and sequence
similarities with exported surface-exposed proteins (PvSTP1, PkSICAvar, PvVIR,
Pf332, and PfEMP1) of several Plasmodium species. SURFIN4.2 of a parasite other
than 3D7 (FCR3S1.2) showed polymorphisms in the extracellular domain, suggesting
sequence variability between genotypes. SURFIN4.2 not only was found
cotransported with PfEMP1 and RIFIN to the IE surface, but also accumulated in
the parasitophorous vacuole. In released merozoites, SURFIN4.2 was present in an
amorphous cap at the parasite apex, where it may be involved in the invasion of
erythrocytes. By exposing shared polymorphic antigens on IEs and merozoites, the
parasite may coordinate the antigenic composition of these attachment surfaces
during growth in the bloodstream. RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of
variant surface antigens (VSA) expressed by infected erythrocytes. VSA
antibodies have been shown to be associated with protection against malaria.
Here, antibody subclass responses to a recombit RIFIN protein (RIF-29) in 116
Ghanaian children were determined by ELISA to investigate the relationship
between severe malaria and anti-RIF-29 antibodies. The study group was composed
of 23 children diagnosed exclusively for cerebral malaria and 35 children who
had non-cerebral severe malaria. The remaining 58 individuals were age-, gender-
and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses
predominated in severe malaria patients compared to matched controls suggests
that these antibodies are not protective, but are most probably induced by a
current infection, an observation substantiated by the equally high reactivity
to both recombit RIF-29 protein and to P. falciparum crude lysate proteins.
The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in
cerebral malaria children brings to mind the possibility that these antibodies
are pathogenic. This is a new finding that may go some way towards explaining
why these children are at risk of developing the life-threatening form of
cerebral malaria. In 1902 Georg Maurer was the first to publish a detailed description of
Giemsa-stained structures in the cytosol of Plasmodium falciparum-infected
erythrocytes, today known as Maurer's clefts. Later when clefts were seen by
electron microscopy, the description was modified to also include these, which
has caused disagreement over the composition of Maurer's clefts. For that
reason, Maurer's clefts were characterized during intraerythrocytic development
of P. falciparum by simultaneously staining cytosolic structures with antibodies
using indirect immunofluorescence assays and with Giemsa. At least three groups
of antigens, P. falciparum erythrocyte membrane protein 1 (PfEMP1)/
RIFIN/SURFIN, P. falciparum histidine-rich protein 2 (PfHRP2), and exported
proteins 1 and 2 (Exp1 and Exp2), were detected in distinct Giemsa-stained
structures in the cytosol of infected erythrocytes, but PfHRP2 and Exp1/Exp2
were not found in clefts by transmission electron microscopy. Therefore,
Maurer's clefts as defined by staining with Giemsa comprise a number of
cytoplasmic structures and antigens not included in structures called clefts and
seen by electron microscopy. In order to avoid immune recognition in favor of a chronic infection, the
malaria parasite Plasmodium falciparum has developed means to express clonally
variant antigens at the surface of the infected erythrocyte (IE). Proteins of
the var and rif multicopy gene families, encoding PfEMP1 and RIFINs,
respectively, have been implicated in these processes. Here, we studied members
of the latter family and present data revealing different subcellular
localization patterns for RIFIN variants belonging to two distinct subgroups,
which have been designated A- and B-type RIFINs. While A-type RIFINs were found
to be associated with the parasite and transported to the surface of infected
erythrocytes via Maurer's clefts, B-type RIFINs appeared to be mostly retained
inside the parasite. However, expression of both subtypes does not seem to be
mutually exclusive. Moreover, both A- and B-type variants were also expressed in
the merozoite, present either in the apical region (A-type) or in the cytosol
(B-type). The presence of RIFINs in merozoites suggests that antigenic variation
in P. falciparum is not only restricted to parasite-derived proteins at the IE
surface, but the phenomenon also prevails in other life cycle stages.
Interestingly, some RIFIN variants were detected only in intracellular stages
and not in merozoites, pointing to differential developmental expression
patterns for distinct members of this large protein family. BACKGROUND: To avoid spleen-dependent killing mechanisms parasite-infected
erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to
bind to endothelial receptors. This binding also known as sequestration, is
mediated by parasite proteins, which are targeted to the erythrocyte surface.
Candidate proteins are those encoded by P. falciparum multicopy gene families,
such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE
sequestration and expression of multicopy gene families is still lacking. Here,
we report on the analysis of IE from a black African immigrant, who received the
diagnosis of a maligt lymphoproliferative disorder and subsequently underwent
splenectomy. Three weeks after surgery, the patient experienced clinical
falciparum malaria with high parasitemia and circulating developmental parasite
stages usually sequestered to the vascular endothelium such as late
trophozoites, schizonts or immature gametocytes.
METHODOLOGY/PRINCIPAL FINDINGS: Initially, when isolated from the patient, the
infected erythrocytes were incapable to bind to various endothelial receptors in
vitro. Moreover, the parasites failed to express the multicopy gene families
var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were
detected. In the course of in vitro cultivation, the parasites started to
express all investigated multicopy gene families and concomitantly developed the
ability to adhere to endothelial receptors such as CD36 and ICAM-1,
respectively.
CONCLUSION/SIGNIFICANCE: This case strongly supports the hypothesis that
parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in
interactions of infected erythrocytes with endothelial receptors mediating
sequestration of mature asexual and immature sexual stages of P. falciparum. In
contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins
may not be involved in sequestration, as these genes were transcribed in
infected but not sequestered erythrocytes. Antibodies to polymorphic antigens expressed during the parasites erythrocytic
stages are important mediators of protective immunity against P. falciparum
malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and
GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine
candidates. However, to what extent these antibodies to blood stage antigens are
acquired during naive individuals' first infections has not been studied in
depth. Using plasma samples collected from controlled experimental P. falciparum
infections we show that antibodies against variant surface antigens, PfEMP1 and
RIFIN as well as MSP3 and GLURP, are acquired during a single short low density
P. falciparum infection in non-immune individuals including strain transcendent
PfEMP1 immune responses. These data indicate that the immunogenicity of the
variant surface antigens is similar to the less diverse merozoite antigens. The
acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies
may reflect a parasite strategy of expressing most or all PfEMP1 variants at
liver release optimizing the likelihood of survival and establishment of chronic
infections in the new host. BACKGROUND: The ability of Plasmodium falciparum to undergo antigenic variation,
by switching expression among protein variants encoded by multigene families,
such as var, rif and stevor, is key to the survival of this parasite in the
human host. The RIFIN protein family can be divided into A and B types based on
the presence or absence of a 25 amino acid motif in the semi-conserved domain. A
particular type B RIFIN, PF13_0006, has previously been shown to be strongly
transcribed in the asexual and sexual stages of P. falciparum in vitro.
METHODS: Antibodies to recombit PF13_0006 RIFIN were used in
immunofluorescence and confocal imaging of 3D7 parasites throughout the asexual
reproduction and sexual development to examine the expression of PF13_0006.
Furthermore, reactivity to recombit PF13_0006 was measured in plasma samples
collected from individuals from both East and West African endemic areas.
RESULTS: The PF13_0006 RIFIN variant appeared expressed by both released
merozoites and gametes after emergence. 7.4% and 12.1% of individuals from East
and West African endemic areas, respectively, carry plasma antibodies that
recognize recombit PF13_0006, where the antibody responses were more common
among older children.
CONCLUSIONS: The stage specificity of PF13_0006 suggests that the diversity of
RIFIN variants has evolved to provide multiple specialized functions in
different stages of the parasite life cycle. These data also suggest that RIFIN
variants antigenically similar to PF13_0006 occur in African parasite
populations. |
What are the current treatments for generalised anxiety disorder in teenagers? | Cognitive-behavioral treatment (CBT) - both in individual and in group treatment
Randomised, placebo controlled trials have found Sertraline efficacious for GAD in adults, children and adolescents.
While both CBT and SSRIs are beneficial, some evidence suggests that the effects of CBT may be more long lasting. | OBJECTIVE: To evaluate the feasibility and effectiveness of a school-based group
cognitive-behavioral treatment (CBT) for anxiety disorders with African-American
adolescents.
METHOD: Twelve adolescents (mean age = 15.6 years) with anxiety disorders were
randomly assigned to CBT (n = 6) or a group attention-support control condition
(AS-Control; n = 6). Both groups met for 10 sessions in the same high school.
Key treatment ingredients in CBT involved exposure, relaxation, social skills,
and cognitive restructuring. Key ingredients in AS-Control involved therapist
and peer support. At pre- and posttreatment, diagnostic interviews were
conducted, and adolescents completed self-report measures of anxiety.
RESULTS: At posttreatment and among those who attended more than one treatment
session, 3/4 adolescents in CBT no longer met diagnostic criteria for their
primary anxiety disorder, compared with 1/5 in AS-Control. Clinician ratings of
impairment and self-report levels of overall anxiety were significantly lower at
posttreatment in CBT compared with AS-Control. Teenagers in both groups reported
lower levels of social anxiety from pre- to posttreatment.
CONCLUSIONS: Findings support the feasibility of implementing a manual-based CBT
in an urban school setting. Responder rates among African-American adolescents
were similar to those found in studies with white youths. BACKGROUND: Family history studies in adults reveal strong familiality for the
anxiety disorders with some specificity. The aim of the current study was to
establish whether there was an elevated rate of anxiety disorders in the parents
of children with anxiety disorders, and whether there was intergenerational
specificity in the form of disorder.
METHODS: The mental state of a clinic sample of 85 children with anxiety
disorder and their parents was systematically assessed, together with a
comparison sample of 45 children with no current disorder and their parents.
RESULTS: Compared to the rate of anxiety disorder amongst parents of comparison
children, the rate of current anxiety disorder in mothers of anxious children
was significantly raised, as was the lifetime rate of anxiety disorder for both
mothers and fathers. The mothers of children with generalised anxiety disorder,
social phobia, specific phobia and separation anxiety disorder all had raised
lifetime rates of the corresponding disorder, but also raised rates of others
disorders.
LIMITATIONS: Only 60% of the fathers of the anxious children were assessed.
CONCLUSIONS: Strong familiality of anxiety disorders was confirmed, especially
between child and maternal anxiety disorder. All child anxiety disorders were
associated with several forms of anxiety disorder in the mother. Some
specificity in the form of anxiety disorder in the child and the mother was
apparent for social phobia and separation anxiety disorder. The findings have
implications for the management of child anxiety. BACKGROUND: In adolescents diagnosed with depressive disorders, psychiatric
comorbidity is rather the rule than the exception.
AIM: To find the prevalence of the association between depression in adolescents
and other psychiatric disorders and second and to study the different mental
disorders comorbid to depression.
METHODS: We conducted a descriptive, retrospective and analytic survey carried
on 77 subjects having been followed in the child psychiatry department of Sfax
for depressive disorders diagnosed according to the DSM-IV TR criteria during a
period of 9 years (from January 1st 1998 till 31st December 2006)
RESULTS: 49.3% of the youths with depression had comorbid conditions: anxiety
disorders in 23.37% of cases, disruptive disorders in 13% of cases (conduct
disorders in 11.7 % of cases and oppositional defiant disorders in 1.3 % of
cases), personality disorders in 13% of cases, substance abuse in 3.9% of cases
and alimentary behavior disturbances in 2.6% of cases. A superimposed major
depressive disorder in adolescents with dysthymia (« Double depression ») was
present in 10.4 of cases.
CONCLUSION: Throughout our study, we underline the frequency of the association
between depression in teenagers and other mental disorders. The detection of
this comorbidity has a great importance as it permits to understand the
pathogenesis of depression in adolescents, to examine the implications of
comorbidity for course and outcomes of this disorder and to elaborate the
appropriate treatment. |
Can tetracycline affect tooth formation? | Tetracycline is incorporated in the teeth during their formation and leads to their permanent staining. A definite relationship between total dosage and staining and duration of administration and staining was established; the condition occurred with greater frequency (in more than one-third of the children) when the total dosage exceeded 3 g. or the duration of treatment was longer than 10 days. | Twenty-four hours after a tetracycline injection, the unimpeded, and more
rapidly erupting, mouse mandibular incisor contained 20% to 44% more
tetracycline than the contralateral, uncut incisor. It was concluded that the
increased tetracycline incorporation reflected a higher rate of mineralization
associated with faster tooth formation in the unimpeded tooth. By measuring the
amount of tetracycline which became incorporated at different times after an
incisor was shortened, it was possible to investigate an early stage of the
response of the incisor to cutting. A significant increase in the capacity of
the tooth to incorporate tetracycline was detectable 4 h after shortening the
tooth, and this was maximal after another 4 h. Complaints of enamel defects in American Indian children residing on the St.
Regis reservation in New York State prompted an epidemiological study. The
results of that study, reported earlier (Rebich et al., 1983), indicated that
over one-fifth of the American Indian children had discoloration of the
dentition due to ingestion of tetracycline during the years of tooth formation.
These data also provided an ideal opportunity to examine the link between
tetracycline staining and caries which has been postulated by previous authors.
American Indian children, ages 7-18, were found to have a higher caries
experience than other children and a lower rate of dental service utilization,
as evidenced by the filled component of the DMFS index (FS/DMFS). Within the
American Indian population, however, no indication was found of any association
between tetracycline staining and dental caries. In this investigation an attempt has been made to determine the relationship
between the staining of permanent teeth by tetracycline administered during the
period of tooth formation with the dosage of the drug and the duration of
therapy. Of 238 subjects whose hospital records indicated ingestion of stated
doses of tetracycline, some 49 were seen to have staining which was confirmed by
fluorescence, and a further six had staining which did not fluoresce and hence
could not be confirmed. A definite relationship between total dosage and
staining and duration of administration and staining was established; the
condition occurred with greater frequency (in more than one-third of the
children) when the total dosage exceeded 3 g. or the duration of treatment was
longer than 10 days. However, as staining was seen at all dosage levels,
whatever the duration, physicians should continue to follow previous advice and
prescribe other antibiotics where possible for children under 8 years of age or
for women in the last trimester of pregcy. Male Wistar rats prelabeled with tetracycline to mark surfaces of bone and tooth
formation-mineralization were placed into orbit for 18.5 days aboard the Soviet
COSMOS-1129 Biosatellite. They were injected with tetracycline for a second and
third time on the 6th and 27th days, respectively, after recovery of the
Biosatellite. Spaceflight did not alter the rate of periosteal bone formation in
the non-weight-bearing ribs and regions of the mandibles, which were covered by
masticatory muscles. Bone formation-calcification rates were impaired at those
sites in the jaw that had no contiguous muscle (molar region). The remodeling
activity on the alveolar bone around the buccal roots of the molar teeth was
significantly reduced but without creating a negative balance between formative
and resorptive activities. Total Ca, P, and hydroxyproline concentrations in the
jaws, incisors, and ribs were normal after spaceflight, but gravity density
fractionation studies indicated that in the jaws alone, O-G conditions caused a
delay in the maturation of bone mineral and matrix. A 29-day postflight recovery
period at earth's gravity was sufficient to fully correct these anomalies.
Relative to tooth formation, relatively normal circadian and infradian
biorhythmic periodicities of Ca and P in dentin and enamel were maintained
during spaceflight. We conclude that most of the non-weight-bearing bones of the
rat skeleton are at risk to the effects of hypogravity. |
Is there any genetic determinant of hair pigmentation that could be useful in forensic analyses? | Yes, there are at least 12 genes associated with human hair color variation such as: TYR, TYRP1, OCA2, SLC45A2, SLC24A5, MC1R, ASIP and KITLG. | We describe a minisequencing protocol for screening DNA samples for the presence
of 12 mutations in the human melanocortin 1 receptor gene (MC1R), eight of which
are associated with the red hair phenotype. A minisequencing profile which shows
homozygosity for one of these mutations or the presence of two different
mutations would strongly indicate that the sample donor is red haired. The
absence of any red hair causing mutations would indicate that the sample donor
does not have red hair. We report the frequencies of MC1R variants in the
British red haired population. Prediction of physical appearance based on genetic analysis is a very attractive
prospect for forensic investigations. Recent studies have proved that there is a
significant association between some genetic variants of the melanocortin 1
receptor (MC1R) gene and red hair color. The present study focuses on the
potential forensic applicability of variation within this pigment-related gene.
Sequencing of the complete MC1R gene was performed on a group of red-haired
individuals and controls with different pigmentation. A major role in
determination of red hair color is played by two MC1R variants--C451T and C478T.
The optimized minisequencing assay for genotyping of the above positions and
three other important red hair-related MC1R polymorphisms, C252A, G425A, and
G880C was successfully applied to analyze typical forensic specimens.
Determination of a homozygous or heterozygous combination can be a good
predictor of both red hair color and fair skin of a subject. The MC1R gene encodes a protein with key regulatory functions in the melanin
synthesis. A multiplex PCR and a multiplex single base extension protocol were
established for genotyping six exonic MC1R variations highly penetrant for red
hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two
frameshift variations highly penetrant for red hair (R) and three variations in
the promoter region. We genotyped 600 individuals from Denmark using either CE
or MALDI-TOF MS as the detection platform. A total of 62 individuals were
genotyped R/R and among the 62 individuals, 57 had red hair and five had blond
hair colour. Two different R alleles may be located in cis (RR/-) position or
trans (R/R) position, and the phenotype associated with RR/- and R/R may be
different. Two allele-specific PCRs were established with primers targeting the
-G445A variation in the MC1R promoter and the allele-specific PCR products were
used in the multiplex single base extension assay. In all 62 individuals, the
MC1R variants were situated in trans position. Another 18 individuals with red
hair colour were either genotyped R/- or R/r, suggesting that other genes
influence hair colour. The natural range of hair and skin colour is a continuous spectrum, controlled
by multiple genes in a complex fashion. Many of these genes are as yet unknown,
but several key pigmentation genes have been characterised, in particular the
melanocortin 1 receptor gene (MC1R). Here, the function and known mutations of
MC1R and other human pigmentation genes including ASIP, MATP, SLC24A5, TYR,
TYRP1 and OCA2 are outlined, and a forensic test based on MC1R SNPs presented.
The forensic utility of this and potential future genetic tests for phenotypic
traits are discussed, in the light of the extensive debate on the ethics of
predicting phenotypic traits from crime scene samples. Human pigmentation is a polygenic trait which may be shaped by different kinds
of gene-gene interactions. Recent studies have revealed that interactive effects
between HERC2 and OCA2 may be responsible for blue eye colour determination in
humans. Here we performed a population association study, examining important
polymorphisms within the HERC2 and OCA2 genes. Furthermore, pooling these
results with genotyping data for MC1R, ASIP and SLC45A2 obtained for the same
population sample we also analysed potential genetic interactions affecting
variation in eye, hair and skin colour. Our results confirmed the association of
HERC2 rs12913832 with eye colour and showed that this SNP is also significantly
associated with skin and hair colouration. It is also concluded that OCA2
rs1800407 is independently associated with eye colour. Finally, using various
approaches we were able to show that there is an interaction between MC1R and
HERC2 in determination of skin and hair colour in the studied population sample. The prediction of an individuals physical appearance from small biological
samples, such as those collected from crime scenes may still sound like science
fiction, but how close are we to achieving this goal? This review provides a
brief introduction to the areas under investigation for direct and indirect
phenotypic inference from DNA alone and suggests some sources of further reading
for those interested in gaining a more in-depth knowledge of this complex
subject. The genetic basis underlying normal variation in the pigmentary traits of skin,
hair and eye colour has been the subject of intense research directed at
understanding the diversity seen both between and within human populations. A
combination of approaches have been used including comparative genomics of
candidate genes and the identification of regions of the human genome under
positive selection, together with genome-wide and specific allele association
studies. Independent selection for different pigmentation gene sets has been
found between Asian, European and African populations. Several genome-wide
association studies for pigmentation have now been conducted and identified
single nucleotide polymorphism (SNP) markers in known, TYR, TYRP1, OCA2,
SLC45A2, SLC24A5, MC1R, ASIP, KITLG and previously unknown SLC24A4, IRF4, TPCN2,
candidate genes. The contribution of SNP polymorphisms present in populations
from South Asia have been tested and alleles found at TYR, SLC45A2 and SLC24A5
can largely account for differences between those of darkest and lightest skin
reflectance using a simple additive model. Skin and hair colour associations in
Europeans are found within a range of pigmentation gene alleles, whereas
blue-brown eye colour can be explained by a single SNP proposed to regulate OCA2
expression. Functional testing of variant alleles has begun to connect phenotype
correlations with biological differences. Variant MC1R alleles show direct
correlations between the biochemical signalling properties of the encoded
receptor and the red-hair fair skin pigmentation phenotype. Direct testing of a
range of clonal melanocyte cultures derived from donor skin tissue characterized
for three causal SNPs within SLC45A2, SLC24A5 and OCA2 has assessed their impact
on melanin content and tyrosinase enzyme activity. From a culmination of genetic
and functional studies, it is apparent that a number of genes impacting
melanosome biogenesis or the melanin biosynthetic pathway are candidates to
explain the diversity seen in human pigmentation. There will always be criminal cases, where the evidence DNA sample will not
match either a suspect's DNA profile, or any in a criminal DNA database. In the
absence of DNA-based mass intelligence screenings, including familial searching
(both of which may be restricted by legislation), there is only one option to
potentially avoid or retrospectively solve "cold cases": the DNA-based
prediction of human externally visible characteristics of an unknown person
based on the crime scene sample left behind. Predictive DNA markers are expected
to be available for some group-specific appearance traits in the near future;
although it is unlikely that we will soon be able to understand the biological
complexity of individual-specific appearance. In suspect-less cases reliable
DNA-based prediction of broader externally visible characteristics from crime
scene samples are expected to reduce the potential pool of suspects by allowing
police investigations to concentrate on specific groups of people. Here, we aim
to describe the forensic motivations for DNA-based prediction of human
externally visible traits as well as the scientific challenges of finding
predictive DNA markers, and will discuss examples with promising (e.g. sex, eye
color and hair color), as well as less promising expectations (e.g. adult body
height), in the foreseen future. Despite the complex ethical and legal
implications arising from DNA-based prediction of externally visible
characteristics, we argue that their use does not lead to a violation of
privacy. We suggest that likelihood-based results, rather than DNA data itself,
should be provided to the police for investigative purposes avoiding data
protection issues. Furthermore, we note that the risk of exacerbating social
pressure on minority groups due to DNA-based prediction of externally visible
traits in crime cases may be reduced rather than increased compared to a
conventional eyewitness testimony. A firm legal basis will need to be
established for the application of these promising qualitative techniques. To
gain the attention of legislative bodies, we invite the forensic community to
participate in a public discourse of these issues. BACKGROUND AND METHODS: Seven genetic biomarkers previously associated with
melanoma were analysed in a meta-analysis conducted in three South European
populations: five red hair colour (RHC) MC1R alleles, one SLC45A2 variant
(p.Phe374Leu) and one thermosensitive TYR variant (p.Arg402Gln). The study
included 1639 melanoma patients and 1342 control subjects.
RESULTS: The estimated odds ratio (OR) associated with carrying at least one
MC1R RHC variant was 2.18 (95% confidence interval (CI): 1.86-2.55;
p-value=1.02×10(-21)), with an additive effect for carrying two RHC variants
(OR: 5.02, 95% CI: 2.88-8.94, p-value=3.91×10(-8)). The SLC45A2 variant,
p.Phe374Leu, was significantly and strongly protective for melanoma in the three
South European populations studied, with an overall OR value of 0.41 (95% CI:
0.33-0.50; p-value=3.50×10(-17)). The association with melanoma of the TYR
variant p.Arg402Gln was also statistically significant (OR: 1.50; 95% CI:
1.11-2.04; p-value=0.0089). Adjustment for all clinical potential confounders
showed that melanoma risks attributable to MC1R and SLC45A2 variants strongly
persisted (OR: 2.01 95% CI: 1.49-2.72 and OR: 0.50, 95% CI: 0.31-0.80,
respectively), while the association of TYR p.Arg402Gln was no longer
significant. In addition, stratification of clinical melanoma risk factors
showed that the risk of melanoma was strong in those individuals who did not
have clinical risk factors.
CONCLUSION: In conclusion, our results show without ambiguity that in South
European populations, MC1R RHC and SCL45A2 p.Phe374Leu variants are strong
melanoma risk predictors, notably in those individuals who would not be
identified as high risk based on their phenotypes or exposures alone. The use of
these biomarkers in clinical practice could be promising and warrants further
discussion. Naturally blond hair is rare in humans and found almost exclusively in Europe
and Oceania. Here, we identify an arginine-to-cysteine change at a highly
conserved residue in tyrosinase-related protein 1 (TYRP1) as a major determit
of blond hair in Solomon Islanders. This missense mutation is predicted to
affect catalytic activity of TYRP1 and causes blond hair through a recessive
mode of inheritance. The mutation is at a frequency of 26% in the Solomon
Islands, is absent outside of Oceania, represents a strong common genetic effect
on a complex human phenotype, and highlights the importance of examining genetic
associations worldwide. In forensic analysis predictive tests for external visible characteristics (or
EVCs), including inference of iris color, represent a potentially useful tool to
guide criminal investigations. Two recent studies, both focused on forensic
testing, have analyzed single nucleotide polymorphism (SNP) genotypes underlying
common eye color variation (Mengel-From et al., Forensic Sci. Int. Genet. 4:323
and Walsh et al., Forensic Sci. Int. Genet. 5:170). Each study arrived at
different recommendations for eye color predictive tests aiming to type the most
closely associated SNPs, although both confirmed rs12913832 in HERC2 as the key
predictor, widely recognized as the most strongly associated marker with blue
and brown iris colors. Differences between these two studies in identification
of other eye color predictors may partly arise from varying approaches to
assigning phenotypes, notably those not unequivocally blue or dark brown and
therefore occupying an intermediate iris color continuum. We have developed two
single base extension assays typing 37 SNPs in pigmentation-associated genes to
study SNP-genotype based prediction of eye, skin, and hair color variation.
These assays were used to test the performance of different sets of eye color
predictors in 416 subjects from six populations of north and south Europe. The
presence of a complex and continuous range of intermediate phenotypes distinct
from blue and brown eye colors was confirmed by establishing eye color
populations compared to genetic clusters defined using Structure software. Our
study explored the effect of an expanded SNP combination beyond six markers has
on the ability to predict eye color in a forensic test without extending the SNP
assay excessively - thus maintaining a balance between the test's predictive
value and an ability to reliably type challenging DNA with a multiplex of
manageable size. Our evaluation used AUC analysis (area under the receiver
operating characteristic curves) and naïve Bayesian likelihood-based
classification approaches. To provide flexibility in SNP-based eye color
predictive tests in forensic applications we modified an online Bayesian
classifier, originally developed for genetic ancestry analysis, to provide a
straightforward system to assign eye color likelihoods from a SNP profile
combining additional informative markers from the predictors analyzed by our
study plus those of Walsh and Mengel-From. Two advantages of the online
classifier is the ability to submit incomplete SNP profiles, a common occurrence
when typing challenging DNA, and the ability to handle physically linked SNPs
showing independent effect, by allowing the user to input frequencies from SNP
pairs or larger combinations. This system was used to include the submission of
frequency data for the SNP pair rs12913832 and rs1129038: indicated by our study
to be the two SNPs most closely associated to eye color. |
What is the suggested clinical management of Fanconi anemia? | Hematopoietic stem cell transplantation is the only proven cure for the hematopoietic manifestations of FA and aggressive lifelong surveillance for solid tumors is essential.In patients with FA, there is a high incidence of aggressive HNSCC at a young age. Surgery remains the mainstay of treatment because patients with FA tolerate radiation therapy and chemotherapy poorly, with significant morbidity | BACKGROUND: Fanconi anemia (FA) is a rare autosomal recessive disorder
characterized by a high degree of genomic instability and predisposition to
cancer development. Recent evidence suggests that the incidence of head and neck
squamous cell carcinoma (HNSCC) may be increased in patients with FA.
OBJECTIVE: To determine the cumulative incidence, tumor distribution, and
outcome of HNSCC in patients with FA.
DESIGN AND SETTING: We analyzed data from 754 subjects from the International
Fanconi Anemia Registry, a prospectively collected database of patients with FA.
MAIN OUTCOME MEASURES: Cumulative incidence of HNSCC and 2-year overall,
relapse-free and disease-specific survival.
RESULTS: Of the 754 patients in the International Fanconi Anemia Registry, 19
(3%) had HNSCC. This is a significantly higher incidence of HNSCC compared with
that observed in the general population (standardized incidence ratio, 500; 95%
confidence interval, 300-781) (P<.001). The patients' age ranged from 15 to 49
years (median, 31 years), and there was a 2:1 female predomice. Surgical
treatment was well tolerated (n = 17); however, radiation therapy and
chemotherapy were associated with significant morbidity and mortality. Of the 19
patients, 10 (53%) developed locoregional recurrences within a median of 16
months from diagnosis. The median follow-up was 29 months. The 2-year
disease-specific, overall, and relapse-free survival rates were 49%, 49%, and
42%, respectively. The cumulative incidence of relapse by the age of 40 years
was 50%.
CONCLUSIONS: In patients with FA, there is a high incidence of aggressive HNSCC
at a young age. Surgery remains the mainstay of treatment because patients with
FA tolerate radiation therapy and chemotherapy poorly, with significant
morbidity. An increased understanding of FA-associated maligcies is not only
important in the clinical management of patients with FA but can also elucidate
the role of chromosomal instability in the development of HNSCC in general. Fanconi anaemia (FA) is an inherited disease with congenital and developmental
abnormalities, bone marrow failure, and extreme risk of leukemic transformation.
Bone marrow surveillance is an important part of the clinical management of FA
and often reveals cytogenetic aberrations. Here, we review bone marrow findings
in FA and discuss the clinical and biological implications of chromosomal
aberrations associated with leukemic transformation. Fanconi anemia (FA) is a heterogeneous disease characterized by spontaneous
chromosomal breaks and abnormal DNA repair. Major clinical problems in FA
include congenital abnormalities, endocrinopathies, early onset bone marrow
failure and increased risk of myelodysplastic syndrome, acute leukemia and solid
tumors. To date, 15 different genes have been shown to cause FA, all of which
have some role in DNA double-strand break repair. Very few strict
genotype-phenotype associations have been identified and clinical manifestations
vary widely from patient to patient, most likely due to modifier genes,
environment and chance effects. Hematopoietic stem cell transplantation is the
only proven cure for the hematopoietic manifestations of FA and aggressive
lifelong surveillance for solid tumors is essential. |
Could Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) cause sudden cardiac death? | Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease that can cause sudden cardiac death. | Cardiac excitation-contraction coupling occurs by a calcium ion-mediated
mechanism in which the signal of action potential is converted into Ca2+ influx
into the cardiomyocytes through the sarcolemmal L-type calcium channels. This is
followed by Ca2+-induced release of additional Ca2+ ions from the lumen of the
sarcoplasmic reticulum into the cytosol via type 2 ryanodine receptors (RyR2).
RyR2 channels form large complexes with additional regulatory proteins,
including FKBP12.6 and calsequestrin 2 (CASQ2). Catecholamines, released into
the body fluids during emotional or physical stress, activate Ca2+-induced Ca2+
release by protein kinase A-mediated phosphorylation of RyR2. Catecholaminergic
polymorphic ventricular tachycardia (CPVT) is an insidious, early-onset and
highly maligt, inherited disorder characterized by effort-induced ventricular
arrhythmias in the absence of structural alterations of the heart. At least some
cases of sudden, unexplained death in young individuals may be ascribed to CPVT.
Mutations of the RyR2 gene cause autosomal domit CPVT, while mutations of the
CASQ2 gene may cause an autosomal recessive or domit form of CPVT. The steps
of the molecular pathogenesis of CPVT are not entirely clear, but inappropriate
"leakiness" of RyR2 channels is thought to play a role; the underlying
mechanisms may involve an increase in the basal activity of the RyR2 channel,
alterations in its phosphorylation status, a defective interaction of RyR2 with
other molecules or ions, such as FKBP12.6, CASQ2, or Mg2+, or its abnormal
activation by extra- or intraluminal Ca2+ ions. Beta-adrenergic antagonists have
proven to be of value in prevention of arrhythmias in CPVT patients, but
occasional treatment failures call for alternative measures. There is great
interest at present for the development of novel antiarrhythmic drugs for CPVT,
as the same approaches may be applied for treatment of more common forms of
life-threatening arrhythmias, such as those arising during ischemia and heart
failure. Sudden cardiac death is defined as an unpredictable death within 24 hours. It is
estimated to occur with a frequency of more than 50,000 per year in Japan. The
inherited arrhythmogenic diseases associated with the transmembranous ionic
channels, anchoring proteins or intracellular calcium regulating proteins are
thought to be responsible for sudden cardiac death in infants, children, and
young adults who have structurally normal hearts. Recent genetic analyses have
identified congenital diseases such as the long-QT syndrome (LQTS), the Jervell
and Lange-Nielsen syndrome (JLNS), the Brugada syndrome (BrS), the short-QT
syndrome (SQTS), the arrhythmogenic right ventricular cardiomyopathy type 2
(ARVC2), and the catecholamine-induced polymorphic ventricular tachycardia
(CPVT) /familial polymorphic ventricular tachycardia (FPVT). Loss of function in
the slow component of the delayed rectifier potassium current (I(Ks)) channels
(KCNQ1, KCNE1), the rapid component of the potassium current (I(Kr)) channels
(KCNH2, KCNE2) and the inward rectifier potassium current (I(Kl), Kir2.1)
channel (KCNJ2) is linked to the LQTSs (type 1, 2, 5, 6, and 7 (Andersen
syndrome)) and the JLNSs (type 1 and 2). Changes of function in the
alpha-subunit of cardiac sodium channels (SCN5A) is also linked to the LQTS type
3 and the BrS. A mutation in the ankyrin-B, anchoring proteins, has been
identified as cause of the LQTS type 4. The SQTS is caused by gain of function
in the KCNH2. Further, the missense mutations in the gene encoding ryanodine
receptor 2 (RyR2) or calsequestrin 2 (CASQ2) that regulate intra-cardiac calcium
handling is possibly implicated in the ARVC2 and the CPVT/FPVT. Herein, we
present a review of the literature regarding the genetic mechanisms of the
inherited arrhythmogenic diseases. The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for
excitation-contraction coupling in the heart and is also present in the brain.
Mutations in RyR2 have been linked to exercise-induced sudden cardiac death
(catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated
RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of
the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the
channel. We found that mice heterozygous for the R2474S mutation in Ryr2
(Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures
(which occurred in the absence of cardiac arrhythmias), exercise-induced
ventricular arrhythmias, and sudden cardiac death. Treatment with a novel
RyR2-specific compound (S107) that enhances the binding of calstabin2 to the
mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac
arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky
Ryr2-R2474S channels in the brain can cause seizures in mice, independent of
cardiac arrhythmias. Based on these data, we propose that CPVT is a combined
neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy,
and the same leaky channels in the heart cause exercise-induced sudden cardiac
death. BACKGROUND: In Europe, sudden cardiac death (SCD) is one of the most common
causes of death. Although sudden cardiac death usually happens in older people,
5% to 10% of the affected individuals are young and apparently healthy. Sudden
death in infants, children, and young adults is relatively rare, with an
incidence of 1 to 5 per 100 000 persons per year. Nonetheless, up to 7000
asymptomatic children die in the USA each year, almost half of them without any
warning signs or symptoms.
METHOD: Selective literature review.
RESULTS: Although structural cardiovascular abnormalities explain most cases of
sudden cardiac death in young people, the cause of death remains unexplained
after autopsy in 10% to 30% of cases. Potentially lethal ion channel disorders
(channelopathies) such as the long QT syndromes (LQTS), catecholaminergic
polymorphic ventricular tachycardia (CPVT), and the Brugada syndrome (BrS) may
account for at least one-third of these unexplained cases. Most of these
diseases are hereditary with autosomal-domit transmission, i.e., there is a
50% chance that the children of affected individuals will be affected
themselves.
CONCLUSIONS: Post-mortem genetic screening for sequence variations in cardiac
ion channel genes has become an important forensic tool for elucidating the
cause of sudden cardiac death. Moreover, it allows the identification of other
family members bearing the previously undiagnosed gene defect, who can then
undergo a cardiological evaluation if indicated by their clinical history. Hereditary non-structural diseases such as catecholaminergic polymorphic
ventricular tachycardia (CPVT), long QT, and the Brugada syndrome as well as
structural disease such as hypertrophic cardiomyopathy (HCM) and arrhythmogenic
right ventricular cardiomyopathy (ARVC) cause a significant percentage of sudden
cardiac deaths in the young. In these cases, genetic testing can be useful and
does not require proxy consent if it is carried out at the request of judicial
authorities as part of a forensic death investigation. Mutations in several
genes are implicated in arrhythmic syndromes, including SCN5A, KCNQ1, KCNH2,
RyR2, and genes causing HCM. If the victim's test is positive, this information
is important for relatives who might be themselves at risk of carrying the
disease-causing mutation. There is no consensus about how professionals should
proceed in this context. This article discusses the ethical and legal arguments
in favour of and against three options: genetic testing of the deceased victim
only; counselling of relatives before testing the victim; counselling restricted
to relatives of victims who tested positive for mutations of serious and
preventable diseases. Legal cases are mentioned that pertain to the duty of
geneticists and other physicians to warn relatives. Although the claim for a
legal duty is tenuous, recent publications and guidelines suggest that
geneticists and others involved in the multidisciplinary approach of sudden
death (SD) cases may, nevertheless, have an ethical duty to inform relatives of
SD victims. Several practical problems remain pertaining to the costs of
testing, the counselling and to the need to obtain permission of judicial
authorities. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac
channelopathy characterized by altered intracellular calcium handling resulting
in ventricular arrhythmias and high risk of cardiac sudden death in young cases
with normal structural hearts. Patients present with exertional syncope and the
trademark dysrhythmia is polymorphic and/or bidirectional ventricular
tachycardia during exercise or adrenergic stimulation. Early detection of CPVT
is crucial because opportune medical intervention prevents sudden cardiac death.
Mutations in the ryanodine receptor RYR2 explain nearly 70% of the CPVT cases
and cause the autosomic domit form of the disease. Mutations in calsequestrin
2 causes a recessive form and explain less than 5% of all cases. Genetic
screening in CPVT, besides providing early detection of asymptomatic carriers at
risk, has provided important insights in the mechanism underlying the disease.
Mutational analysis of RYR2 has been a challenge due to the large size of the
gene, 105 exons encoded for 4,967 amino-acids. In this review we analyze general
concepts of the disease, differential diagnosis and strategies for genetic
screening. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic disease that can cause sudden cardiac death due to ventricular
fibrillation (VF). While pharmacological therapy with beta-blockers and/or
Ca(2)(+) antagonists is often unreliable, a recent study has demonstrated that
flecainide can effectively suppress arrhythmia in a murine model of CPVT as well
as clinically in two human subjects suffering from CPVT. We here present the
case of an 11-year-old boy suffering from CPVT-1 as well as a review of the
current relevant literature. After resuscitation due to VF at age 9, an
automated implantable cardioverter-defibrillator (ICD) was implanted in 2007.
Under beta-blocker therapy, repeated shocks were delivered due to either fast
ventricular tachycardia (VT) or VF. This persisted under additional therapy with
verapamil. Implantable cardioverter-defibrillator routine interrogations showed
frequent non-sustained VT with an average of 8.8 per day. Additionally, the
patient suffered from impaired physical performance due to decreased
chronotropic competence. In July 2009, flecainide was added to the
beta-blocker/verapamil regimen, resulting in a plasma level of 0.20 mg/L. No ICD
shock or sustained VT occurred until December 2010. Genetic testing revealed an
RyR2 receptor mutation. The case demonstrates the challenge of diagnosis and
management of CPVT. It furthermore supports recent experimental evidence that
the class 1 antiarrhythmic drug flecainide can suppress CPVT. The presented case
supports a novel strategy in treating CPVT with the class I antiarrhythmic agent
flecainide. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare
adrenergically mediated arrhythmogenic disorder classically induced by exercise
or emotional stress and found in structurally normal hearts. It is an important
cause of cardiac syncope and sudden death in childhood. Catecholaminergic
polymorphic ventricular tachycardia is a genetic cardiac channelopathy with
known mutations involving genes affecting intracellular calcium regulation. We
present a case of a 14-year-old boy who had cardiopulmonary arrest after an
emotionally induced episode of CPVT while attempting to invite a girl to the
school dance. Review of his presenting cardiac rhythm, induction of concerning
ventricular arrhythmias during an exercise stress test, and genetic testing
confirmed the diagnosis of CPVT. He recovered fully and was treated with
β-blocker therapy and placement of an implantable cardioverter-defibrillator. In
this report, we discuss this rare but important entity, including its molecular
foundation, clinical presentation, basics of diagnosis, therapeutic options, and
implications of genetic testing for family members. We also compare CPVT to
other notable cardiomyopathic and channelopathic causes of sudden death in youth
including hypertrophic cardiomyopathy, arrhythmogenic right ventricular
dysplasia, long QT syndrome, short QT syndrome, and Brugada syndrome. Sudden cardiac death (SCD) is one of the most common causes of death in
developed countries, with most SCDs involving the elderly, and structural heart
disease evident at autopsy. Each year, however, thousands of sudden deaths
involving individuals younger than 35 years of age remain unexplained after a
comprehensive medicolegal investigation that includes an autopsy. In fact,
several epidemiologic studies have estimated that at least 3% and up to 53% of
sudden deaths involving previously healthy children, adolescents, and young
adults show no morphologic abnormalities identifiable at autopsy. Cardiac
channelopathies associated with structurally normal hearts such as long QT
syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT),
and Brugada syndrome (BrS) yield no evidence to be found at autopsy, leaving
coroners, medical examiners, and forensic pathologists only to speculate that a
lethal arrhythmia might lie at the heart of a sudden unexplained death (SUD). In
cases of autopsy-negative SUD, continued investigation through either a
cardiologic and genetic evaluation of first- or second-degree relatives or a
molecular autopsy may elucidate the underlying mechanism contributing to the
sudden death and allow for identification of living family members with the
pathogenic substrate that renders them vulnerable, with an increased risk for
cardiac events including syncope, cardiac arrest, and sudden death. In this study, the clinical and implantable cardioverter-defibrillator
(ICD)-related follow-up of patients with catecholaminergic polymorphic
ventricular tachycardia (CPVT) with homogenous missense mutations in CASQ2 was
summarized. Patients were followed in a pediatric cardiology clinic and an ICD
clinic. All patients were treated with high-dose β blockers. ICDs were
recommended for patients who remained symptomatic despite medical treatment.
Twenty-seven patients were followed for 1 to 15 years (median 9). Twenty
patients (74%) were symptomatic at diagnosis; 13 (65%) remained symptomatic
after treatment with high-dose β blockers and thus were advised to receive ICDs.
Eight of these patients refused ICDs, and eventually 6 (75%) died suddenly. Four
of the 5 patients who received ICDs had ventricular tachycardia storms treated
but not terminated by recurrent ICD shocks. These ventricular tachycardia storms
(2 episodes in 2 patients and 1 episode in 2 patient) terminated spontaneously
after finishing the programmed ICD shocks, without degeneration to ventricular
fibrillation. None of the patients who received ICDs died. In conclusion,
patients with CASQ2-associated CPVT should be recommended to receive ICDs to
prevent sudden death when medical therapy is not effective. These patients may
have recurrent ventricular tachycardia storms treated but not terminated by
recurrent ICD shocks, without degeneration to ventricular fibrillation. BACKGROUND: Sudden cardiac death of a child is a devastating event for the
family and an enormous challenge for the attending physician.
METHODS AND RESULTS: We report a family with repeat events of sudden cardiac
death and recurrent ventricular fibrillation in a teenage girl, where autopsy
data and clinical investigations were inconclusive. The diagnosis of
catecholaminergic polymorphic ventricular tachycardia (CPVT) was established
only following finding a gene mutation in the cardiac ryanodine receptor.
CONCLUSIONS: Interpretation of autopsy data, provocation testing and genetic
testing in victims of sudden death and family members are discussed to correctly
identify the cause and properly manage asymptomatic carriers in such families. Each year infants, children and young adults die suddenly and unexpectedly. In
many cases the cause of death can be elucidated by medico-legal autopsy,
however, a significant number of these cases remain unexplained despite a
detailed postmortem investigation and are labeled as sudden unexplained death
(SUD). Post-mortem genetic testing, so called molecular autopsy, revealed that
primary arrhythmogenic disorders including long QT syndrome and
catecholaminergic polymorphic ventricular tachycardia (CPVT) may account for a
certain number of these cases. Because of the inheritance of these diseases,
close relatives of the deceased may also at potential risk of carrying fatal
cardiac disorders. Therefore, advanced diagnostic analyses, genetic counseling
and interdisciplinary collaboration should be integral parts of clinical and
forensic practice. In the present study, we performed mutation analyses of the
major genes causing cardiac channelopathies in 15 SUD cases. In four cases we
found putative pathogenic mutations in cardiac ion channel genes. Clinical and
genetic examination of family members of SUD victims was also performed and
affected family members were identified. This study demonstrates that molecular
genetic screening needs to become an inherent part of the postmortem
examination. This will enhance the ability of screening family members of SUD
victims who may be at risk. The present data also illustrate that detection and
follow up of familial cases of sudden death is challenging and requires a close
multidisciplinary collaboration between different medical disciplines, with
great responsibility for the forensic pathologist. 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. SADS is defined as sudden death under the age of 40 years old in the absence of
structural heart disease. Family screening studies are able to identify a cause
in up to 50% of cases-most commonly long QT syndrome (LQTS), Brugada and early
repolarization syndrome, and catecholaminergic polymorphic ventricular
tachycardia (CPVT) using standard clinical screening investigations including
pharmacological challenge testing. These diagnoses may be supported by genetic
testing which can aid cascade screening and may help guide management. In the
current era it is possible to undertake molecular autopsy provided suitable
samples of DNA can be obtained from the proband. With the evolution of rapid
sequencing techniques it is possible to sequence the whole exome for candidate
genes. This major advance offers the opportunity to identify novel causes of
lethal arrhythmia but also poses the challenge of managing the volume of data
generated and evaluating variants of unknown significance (VUS). The emergence
of induced pluripotent stem cell technology could enable evaluation of the
electrophysiological relevance of specific ion channel mutations in the proband
or their relatives and will potentially enable screening of idiopathic
ventricular fibrillation survivors combining genetic and electrophysiological
studies in derived myocytes. This also could facilitate the assessment of
personalized preventative pharmacological therapies. This review will evaluate
the current screening strategies in SADS families, the role of molecular autopsy
and genetic testing and the potential applications of molecular and cellular
diagnostic strategies on the horizon. BACKGROUND: Distinguishing genetic variants that cause disease from variants
that are rare but benign is one of the principal challenges in contemporary
clinical genetics, particularly as variants are identified at a pace exceeding
the capacity of researchers to characterise them functionally.
METHODS: We previously developed a novel method, called paralogue annotation,
which accurately and specifically identifies disease-causing missense variants
by transferring disease-causing annotations across families of related proteins.
Here we refine our approach, and apply it to novel variants found in 2266
patients across two large cohorts with inherited sudden death syndromes, namely
catecholaminergic polymorphic ventricular tachycardia (CPVT) or Brugada syndrome
(BrS).
RESULTS: Over one third of the novel non-synonymous variants found in these
studies, which would otherwise be reported in a clinical diagnostics setting as
'variants of unknown significance', are categorised by our method as likely
disease causing (positive predictive value 98.7%). This identified more than 500
new disease loci for BrS and CPVT.
CONCLUSIONS: Our methodology is widely transferable across all human disease
genes, with an estimated 150 000 potentially informative annotations in more
than 1800 genes. We have developed a web resource that allows researchers and
clinicians to annotate variants found in individuals with inherited arrhythmias,
comprising a referenced compendium of known missense variants in these genes
together with a user-friendly implementation of our approach. This tool will
facilitate the interpretation of many novel variants that might otherwise remain
unclassified. |
Does Apolipoprotein E (ApoE) have anti-inflammatory activity? | Yes. ApoE has anti-inflammatory activity | PAF and PAF-like oxidized phospholipids hydrolysed by platelet-activating factor
(PAF) acetylhydrolase (AH) are potent lipid mediators involved in inflammation
and atherosclerosis. Apolipoprotein (apo) E-containing high-density lipoprotein
(HDL) has antioxidant, anti-inflammatory and anti-atherogenic properties. The
study investigated apoE-containing HDL-associated PAF-AH (HDL-PAF-AH) and total
(apoE-containing+apoE-poor) HDL-PAF-AH activities as well as malondialdehyde
(MDA) concentration in 291 patients with polycystic ovary syndrome (PCOS) using
the Rotterdam consensus criteria and 281 control women. Compared with the
control women, patients with hyperandrogenism+oligo/anovulation+polycystic
ovaries (PCO) or hyperandrogenism+PCO had lower total, apoE-containing and
apoE-poor HDL-PAF-AH activities, while those with oligo/anovulation+PCO showed
decreased total and apoE-poor HDL-PAF-AH activities. Other factors including
insulin resistance and obesity in PCOS had the adverse effects associated with
the HDL-PAF-AH activities. Serum MDA concentration was associated with PCOS,
hyperandrogenism, insulin resistance and hypertriglyceridaemia in patients with
PCOS. Decreased total and apoE-containing HDL-PAF-AH activities and increased
serum MDA concentration may contribute to the pathogenesis of PCOS and
potentially link to related complications responsible for oxidative stress and
inflammation such as an increased risk for type 2 diabetes mellitus and/or
future cardiovascular diseases in PCOS patients. BACKGROUND: After age, the second largest risk factor for Alzheimer's disease
(AD) is apolipoprotein E (APOE) genotype, where APOE4 is associated with lower
apoE protein levels, more severer brain pathology, enhanced inflammation and
disease. Small peptides corresponding to the receptor-binding region of apoE
mimic the anti-inflammatory activity of the apoE holoprotein. These apoE
mimetics greatly improve behavioral outcomes and neuronal survival in head
trauma models that display AD pathology and neuronal loss.
OBJECTIVE: To determine whether apoE mimetics change behavior, inflammation and
pathology in CVND-AD (SwDI-APP/NOS2(-/-)) transgenic mice.
METHODS: Starting at 9 months, apoE peptides were subcutaneously administered 3
times per week for 3 months followed by behavioral, histochemical and
biochemical testing.
RESULTS: Treatment with apoE mimetics significantly improved behavior while
decreasing the inflammatory cytokine IL-6, neurofibrillary tangle-like and
amyloid plaque-like structures. Biochemical measures matched the visible
pathological results.
CONCLUSIONS: Treatment with apoE mimetics significantly improved behavior,
reduced inflammation and reduced pathology in CVND-AD mice. These improvements
are associated with apoE-mimetic-mediated increases in protein phosphatase 2A
activity. Testing in additional AD models showed similar benefits, reinforcing
this novel mechanism of action of apoE mimetics. These data suggest that the
combination of anti-inflammatory and neuroprotective activities of apoE mimetics
represents a new generation of potential therapeutics for AD. |
Which drugs acting via bradykinin system are effective for treatment of ACE-inhibitor-induced angioedema? | Icatibant and ecallantide are medication acting via bradykinin system that are used for treatment of ACE-inhibitor-induced angioedema. | Angiotensin-converting enzyme (ACE) inhibitors block the catalysis of
angiotensin I to angiotensin II and also the breakdown of bradykinin. ACE
inhibitor-induced angioedema is mediated by inhibited bradykinin degradation
leading to enhanced bradykinin plasma levels. The efficacy of currently used
standard treatments with antiallergic drugs is questionable. A patient with
acute ACE inhibitor-induced angioedema was treated with icatibant, a specific
bradykinin B2 receptor antagonist approved for the treatment of hereditary
angioedema. A single subcutaneous injection of 30 mg icatibant resulted in a
rapid onset of symptom relief and a remarkable shortening of duration of the
attack. Angiotensin-converting enzyme inhibitors (ACEI) are commonly prescribed for
blood pressure control and renal protection. ACEI angioedema is a common problem
in patients who are taking ACEI, although, in most cases, the disorder is
self-limited, and spontaneous episodes of apparently unprovoked angioedema stop
with the discontinuation of the medication. In a subset of patients,
hospitalization and even intubation are required for airway protection. The
diagnosis is made clinically. There are no laboratory studies that establish the
diagnosis. However, such investigations help exclude alternative diagnoses as
the cause for the patient's presentation. Conventional treatment with regimens
used to control allergic angioedema is ineffective in this condition. The
mechanism of ACEI-induced angioedema is thought to be related to its effect on
the kallikrein-kinin system. Kallikrein is a protease that converts
high-molecular-weight kininogens into kinins, primarily bradykinin. Medications
recently developed, primarily icatibant and ecallantide, to control hereditary
angioedema, a disorder also associated with kallikrein-kinin activation, have
been used to treat ACEI angioedema with some success. The efficacy of these
agents and their optimal use remains to be established by randomized and placebo
controlled trials. |
Is the ACE inhibitor indicated for lung cancer treatment? | No, the angiotensin converting enzyme (ACE) inhibitors are used widely as antihypertensive agents. On the contrary, it has been suggested that they decrease the risk of some cancers, although available data are conflicting. One study proposes that captopril could be a promising option for the treatment of lung cancer. Furthermore, angiotensin-converting enzyme (ACE) inhibitors have been shown to mitigate radiation-induced lung injury in preclinical models | PURPOSE: Angiotensin-converting enzyme (ACE) inhibitors have been shown to
mitigate radiation-induced lung injury in preclinical models. The aim of this
study was to evaluate whether ACE inhibitors decrease the risk of radiation
pneumonitis in lung cancer patients receiving thoracic irradiation.
METHODS AND MATERIALS: Patients with Stage I through III small-cell and
non-small-cell lung cancer treated definitively with radiation from 2004-2009 at
the Clement J. Zablocki Veterans Affairs Medical Center were retrospectively
reviewed. Acute pulmonary toxicity was quantified within 6 months of completion
of treatment according to the Common Terminology Criteria for Adverse Events
version 4. The use of ACE inhibitors, nonsteroidal anti-inflammatory drugs,
inhaled glucocorticosteroids, statins, and angiotensin receptor blockers;
dose-volume histogram parameters; and patient factors were assessed for
association with Grade 2 or higher pneumonitis.
RESULTS: A total of 162 patients met the criteria for inclusion. The majority of
patients had Stage III disease (64%) and received concurrent chemotherapy (61%).
Sixty-two patients were identified as ACE inhibitor users (38%). All patients
had acceptable radiation plans based on dose-volume histogram constraints (V20
[volume of lung receiving at least 20 Gy] ≤37% and mean lung dose ≤20 Gy) with
the exception of 2 patients who did not meet both criteria. Grade 2 or higher
pulmonary toxicity occurred in 12 patients (7.4%). The rate of Grade 2 or higher
pneumonitis was lower in ACE inhibitor users vs. nonusers (2% vs. 11%, p =
0.032). Rates of Grade 2 or higher pneumonitis were significantly increased in
patients aged greater than 70 years (16% vs. 2%, p = 0.005) or in whom V5
(volume of lung receiving at least 5 Gy) was 50% or greater (13% vs. 4%, p =
0.04). V10 (volume of lung receiving at least 10 Gy), V20, V30 (volume of lung
receiving at least 30 Gy), and mean lung dose were not independently associated
with Grade 2 or higher pneumonitis.
CONCLUSION: ACE inhibitors may decrease the incidence of radiation pneumonitis
in patients receiving thoracic radiation for lung cancer. These findings are
consistent with preclinical evidence and should be prospectively evaluated. |
Which forms of cancer is the Tpl2 gene associated with? | Tpl2/Map3K8, also known as tumor progression locus 2 has been identified as an oncogene, its mutation or overexpression is reported in a variety of human cancers. Types of cancer associated with Tpl2 include skin and epithelial cancers, ADI prostate cancer, gastric and colon adenocarcinomas, colitis-associated cancer (CAC), breast cancer, Hodgkin lymphomas, nasopharyngeal carcinomas and several types of T-cell neoplasias. | BACKGROUND: Despite its initial positive response to hormone ablation therapy,
prostate cancers invariably recur in more aggressive, treatment resistant forms.
The lack of our understanding of underlying genetic alterations for the
transition from androgen-dependent (AD) to ADI prostate cancer growth hampers
our ability to develop target-driven therapeutic strategies for the efficient
treatment of ADI prostate cancer.
METHODOLOGY/PRINCIPAL FINDINGS: By screening a library of activated human
kinases, we have identified TPL2, encoding a serine/threonine kinase, as driving
ADI prostate cancer growth. TPL2 activation by over-expressing either wild-type
or a constitutively activated form of TPL2 induced ADI growth, whereas the
suppression of TPL2 expression and its kinase activity in ADI prostate cancer
cells inhibited cell proliferation under androgen-depleted conditions. Most
importantly, TPL2 is upregulated in ADI prostate cancers of both the Pten
deletion mouse model and the clinical prostate cancer specimens.
CONCLUSIONS/SIGNIFICANCE: Together these data suggest that TPL2 kinase plays a
critical role in the promotion of ADI prostate cancer progression. Furthermore,
the suppression of TPL2 diminishes ADI prostate cancer growth and a high
frequency of TPL2 overexpression in human ADI prostate cancer samples validates
TPL2 as a target for the treatment of this deadly disease. To address the role of Tpl2, a MAP3K8 that regulates innate/adaptive immunity
and inflammation, in intestinal tumorigenesis, we crossed a Tpl2 KO allele into
the Apc(min/+) genetic background. Here, we show that Apc(min/+)/Tpl2(-/-) mice
exhibit a fivefold increase in the number of intestinal adenomas. Bone marrow
transplantation experiments revealed that the enhancement of polyposis was
partially hematopoietic cell-driven. Consistent with this observation, Tpl2
ablation promoted intestinal inflammation. IL-10 levels and regulatory T-cell
numbers were lower in the intestines of Tpl2(-/-) mice, independent of Apc and
polyp status, suggesting that they were responsible for the initiation of the
enhancement of tumorigenesis caused by the ablation of Tpl2. The low IL-10
levels correlated with defects in mTOR activation and Stat3 phosphorylation in
Toll-like receptor-stimulated macrophages and with a defect in inducible
regulatory T-cell generation and function. Both polyp numbers and inflammation
increased progressively with time. The rate of increase of both, however, was
more rapid in Apc(min/+)/Tpl2(-/-) mice, suggesting that the positive feedback
initiated by inflammatory signals originating in developing polyps is more
robust in these mice. This may be because these mice have a higher intestinal
polyp burden as a result of the enhancement of tumor initiation. |
What are the indications for hydrochlorothiazide? | Hydrochlorothiazide is a diuretic, often used in combination with others. Hydrochlorothiazide are used to treat hypertension. Hydrochlorothiazide has been shown to decrease diastolic blood pressure. | BACKGROUND: Cardiovascular disease (CVD) places a significant burden on
healthcare providers. High blood pressure (BP) is the single most prevalent risk
factor for CVD worldwide and is responsible for more deaths than any other risk
factor. 'Cardiovascular (CV) high-risk patients' make up the broad cross-section
of patients in the middle of the risk spectrum for CVD progression that is
referred to as the CV continuum and includes those with atherothrombotic
disease, those with target organ damage associated with type 2 diabetes and
those with multiple risk factors. Angiotensin II is involved in CVD progression
at every stage of the CV continuum, making the renin-angiotensin system a
rational target for pharmacologic intervention. Angiotensin II receptor blockers
(ARBs) offer a better tolerated alternative to angiotensin converting enzyme
inhibitors, with greater long-term adherence. The ARB telmisartan recently
received an indication for CV prevention.
SCOPE: A PubMed literature search was conducted to identify evidence on the use
of telmisartan for preventing CV events.
FINDINGS: Telmisartan has a favourable safety and tolerability profile, and has
demonstrated efficacious and long-lasting 24-hour BP reductions, whether as
monotherapy or in combination with hydrochlorothiazide or amlodipine. In the
largest CV prevention trial program undertaken with an ARB (the ONgoing
Telmisartan Alone and in combination with Ramipril Global Endpoint Trial;
ONTARGET), telmisartan 80 mg/day alone was as effective as ramipril in reducing
the composite primary endpoint of CV mortality, non-fatal myocardial infarction,
non-fatal stroke and hospitalization for heart failure in CV high-risk patients.
However, patients were significantly more likely to adhere to treatment with
telmisartan than ramipril due to its better tolerability.
CONCLUSION: To date, telmisartan is the only ARB indicated to reduce CV
morbidity in a broad CV high-risk population. This 16-week trial investigated the efficacy and safety of single-pill
valsartan/hydrochlorothiazide (HCTZ) vs. the individual components in patients
70 years and older with systolic hypertension. Patients were randomized to
valsartan/HCTZ 160/12.5 mg (n=128), HCTZ 12.5 mg (n=128), or valsartan 160 mg
(n=128) for 4 weeks. Patients whose blood pressure (BP) was ≥140/90 mm Hg at
weeks 4, 8, or 12 were up-titrated to a maximum of valsartan/HCTZ 320/25 mg.
Week 4 systolic BP reduction (primary efficacy outcome) was greater with
valsartan/HCTZ than valsartan (-17.3 mm Hg vs. -8.6 mm Hg, P <.0001) but only
marginally greater than HCTZ (-13.6 mm Hg, P =.096). Median time to BP control
was shorter with valsartan/HCTZ (4 weeks) vs HCTZ (8 weeks, P<.05) or valsartan
(12 weeks, P<.0001). Thiazide monotherapy was more effective than angiotensin
receptor blocker monotherapy (by about 5 mm Hg), but greater antihypertensive
efficacy was achieved by initiating treatment with combination valsartan/HCTZ in
the elderly. Hypertension has a major associated risk for organ damage and mortality, which
is further heightened in patients with prior cardiovascular (CV) events,
comorbid diabetes mellitus, microalbuminuria and renal impairment. Given that
most patients with hypertension require at least two antihypertensives to
achieve blood pressure (BP) goals, identifying the most appropriate combination
regimen based on individual risk factors and comorbidities is important for risk
management. Single-pill combinations (SPCs) containing two or more
antihypertensive agents with complementary mechanisms of action offer potential
advantages over free-drug combinations, including simplification of treatment
regimens, convenience and reduced costs. The improved adherence and convenience
resulting from SPC use is recognised in updated hypertension guidelines. Despite
a wide choice of SPCs for hypertension treatment, clinical evidence from direct
head-to-head comparisons to guide selection for individual patients is lacking.
However, in patients with evidence of renal disease or at greater risk of
developing renal disease, such as those with diabetes mellitus, microalbuminura
and high-normal BP or overt hypertension, guidelines recommend renin-angiotensin
system (RAS) blocker-based combination therapy due to superior renoprotective
effects compared with other antihypertensive classes. Furthermore, RAS
inhibitors attenuate the oedema and renal hyperfiltration associated with
calcium channel blocker (CCB) monotherapy, making them a good choice for
combination therapy. The occurrence of angiotensin-converting enzyme (ACE)
inhibitor-induced cough supports the use of angiotensin II receptor blockers
(ARBs) for RAS blockade rather than ACE inhibitors. In this regard, ARB-based
SPCs are available in combination with the diuretic, hydrochlorothiazide (HCTZ)
or the calcium CCB, amlodipine. Telmisartan, a long-acting ARB with preferential
pharmacodynamic profile compared with several other ARBs, and the only ARB with
an indication for the prevention of CV disease progression, is available in two
SPC formulations, telmisartan/HCTZ and telmisartan/amlodipine. Clinical studies
suggest that in CV high-risk patients and those with evidence of renal disease,
the use of an ARB/CCB combination may be preferred to ARB/HCTZ combinations due
to superior renoprotective and CV benefits and reduced metabolic side effects in
patients with concomitant metabolic disorders. However, selection of the most
appropriate antihypertensive combination should be dependent on careful review
of the individual patient and appropriate consideration of drug pharmacology. Hypertension affects approximately 26% of the world's adult population and is a
recognized major risk factor for morbidity and mortality associated with
cardiovascular, cerebrovascular, and renal diseases. However, despite the
availability of a range of effective antihypertensive agents and a growing
awareness of the consequences of high blood pressure (BP), the treatment and
control of hypertension remains suboptimal. A number of patient subgroups are
categorized as 'high risk' and may have hypertension that is more difficult to
treat, including obese individuals, patients with stage 2 hypertension, those
with type 2 diabetes mellitus (T2DM), patients with coronary artery disease or a
history of stroke, and Black patients. As the benefits of lowering BP in
patients with hypertension are unequivocal, particularly in high-risk patients,
treating high-risk patients with hypertension to BP goals and maintaining
24-hour BP control is important to help reduce cardiovascular risk and improve
outcomes. Although the BP goals recommended in current consensus guidelines for
the management of patients with hypertension are based on cuff BP measurements,
ambulatory BP monitoring (ABPM) provides a valuable diagnostic tool and allows a
more accurate assessment of BP levels throughout the 24-hour dosing period. ABPM
is a better predictor of prognosis than office BP measurement and is also useful
for assessing whether antihypertensive therapy remains effective in the critical
last few hours of the dosing period, which usually coincides with the morning BP
surge associated with arousal and arising. ABPM has been adopted by new
evidence-based guidelines in the United Kingdom to confirm a suspected diagnosis
of hypertension, which is an indication of the growing importance of ABPM in the
management of hypertension. This review provides an overview of the efficacy and
safety of antihypertensive therapy based on olmesartan
medoxomil ± hydrochlorothiazide and amlodipine/olmesartan medoxomil in high-risk
patient populations enrolled in studies that reported ambulatory BP endpoints.
The studies identified in this review showed that a titrate-to-BP goal strategy
using olmesartan medoxomil- or amlodipine/olmesartan medoxomil-based
antihypertensive therapy was an effective and well-tolerated approach for
maintaining BP control throughout the full 24-hour dosing period in high-risk
patients with difficult-to-treat hypertension. OBJECTIVE: To determine whether thiazides have a chronic antihypertensive
effect, in the absence of diuresis, in patients with severe renal disease
(creatinine clearance <30 mL/min) or in those receiving dialysis.
DATA SOURCES: A search was performed in PubMed, CENTRAL, and International
Pharmaceutical Abstracts, using MeSH terms and/or key words. MeSH terms included
kidney failure, chronic and exploded terms hydrochlorothiazide, renal dialysis,
and thiazides. Key words included thiazide*, hydrochlorothiazide,
chlorothiazide, chlorthalidone, indapamide, metolazone, methyclothiazide,
bendroflumethiazide, hemodialysis, dialysis, kidney failure, renal failure,
renal insufficiency, hypertension, vasodilation, vascular, and diuretics.
STUDY SELECTION AND DATA EXTRACTION: All relevant English-language publications
were evaluated. Studies evaluating the efficacy of thiazides in renal
insufficiency or dialysis were limited to those that included blood pressure
measurements. Studies were included only if treatment duration was at least 4
weeks to evaluate chronic antihypertensive effects.
DATA SYNTHESIS: Thiazide diuretics are associated with a chronic reduction in
peripheral vascular resistance secondary to a purported vasodilatory effect.
However, few clinical studies have evaluated the chronic antihypertensive
efficacy of thiazide and thiazide-like diuretics in patients with severe renal
disease or those on dialysis. Agents studied include hydrochlorothiazide,
chlorothiazide, indapamide, and metolazone, with results varying by drug and
patient population. Hydrochlorothiazide 25-200 mg daily, chlorothiazide 500 mg
twice daily, and indapamide 2.5 mg daily provided long-term blood pressure
reduction in patients with severe renal disease who were not on dialysis. In
studies involving patients on dialysis, hydrochlorothiazide 50 mg daily and
metolazone 5 mg daily did not affect blood pressure; however, 1 study suggested
that indapamide 2.5 mg daily may confer an antihypertensive effect. All studies
were small (≤12 subjects) and had methodological limitations.
CONCLUSIONS: Thiazide diuretics may decrease peripheral vascular resistance
independent of natriuresis. However, because current clinical data are
inconclusive as to the efficacy of these agents at chronically lowering blood
pressure in patients with severe renal disease or in those on dialysis, thiazide
diuretics cannot be routinely recommended for this indication. The article gives an overview of the risk factors for hypertension and the
appropriate indication for using a fixed combination of telmisartan and
hydrochlorothiazide. It cites large, multicentre clinical trials that
demonstrate the efficacy and safety of telmisartan in patients with hypertension
and also a metaanalysis of treatment with either telmisartan alone (TELMI) or in
combination with hydrochlorothiazide (HCTZ) which shows that it is a very well
tolerated and safe treatment for patients across a wide age range. Diuretics belong to the basic group of medicines for the treatment of
hypertension and heart failure. In the case of hypertension treatment, their
main indication is higher age and isolated systolic hypertension. In the case of
heart failure they are used for the treatment of swellings and shortness of
breath. The most frequently prescribed group of diuretics is thiazides and
similar products. In patients with renal insufficiency, loop diuretics are
administered. In the case of hypertension, diuretics are mainly used in the
combination treatment. The most frequently used diuretic in combination is again
hydrochlorothiazide, which is combined with renigiotensin system blockers. It
is mainly the combination of an ACE inhibitor + indapamide that seems to be
modern and promising, and it is, on the basis of large clinical trials,
recommended also for diabetics (ADVANCE) or for secondary prevention following a
cerebrovascular accident (PROGRESS) or for the elderly (HYVET). Also a
combination of two diuretics is popular - mainly hydrochlorothiazide +
amiloride. A combination of a betablocker and diuretic is less suitable. |
Are genes symmetrically distributed between leading and lagging DNA strand in bacteria? | In most bacteria, genes are preferentially encoded on the leading strand than on the lagging strand. This avoids the potentially detrimental head-on collisions that occur between the replication and transcription machineries when genes are encoded on the lagging strand. Head-on collisions are more deleterious than codirectional collisions, and may lead to replication fork arrest and genomic instability. Genes of some functional categories such as ribosome have higher preferences to be on the leading strands, while genes of other functional categories such as transcription factor have higher preferences on the lagging strands. Strand-biased gene distribution correlates with replication-associated purine asymmetry and the presence or absence of polC. Especially essential and highly transcribed genes and genes whose expression is important for fitness are more preferentially situated at the leading strand in bacteria. | We have elaborated a method which has allowed us to estimate the direction of
translocation of orthologs which have changed, during the phylogeny, their
positions on chromosome in respect to the leading or lagging role of DNA
strands. We have shown that the relative number of translocations which have
switched positions of genes from the leading to the lagging DNA strand is lower
than the number of translocations which have transferred genes from the lagging
strand to the leading strand of prokaryotic genomes. This paradox could be
explained by assuming that the stronger mutation pressure and selection after
inversion preferentially eliminate genes transferred from the leading to the
lagging DNA strand. Replication generates bacterial chromosomes with strands that differ in the
number of genes and base composition. It has been suggested that in bacteria
such as Bacillus subtilis, PolC is responsible for the synthesis of the leading
strand and DnaE for the lagging strand, whereas in many other bacteria DnaE is
responsible for the synthesis of both strands. Here, I show that the possession
of PolC correlates with leading strands that contain an average of 78% of genes
compared with 58% for genomes that do not contain PolC. This suggests that
asymmetrical replication forks could have a major role in defining and
constraining the structure of the bacterial chromosome. The presence of PolC is
not correlated with compositional strand bias, suggesting that the two biases
result from different types of structural asymmetry. Many bacterial genomes are under asymmetric mutational pressure which introduces
compositional asymmetry into DNA molecule resulting in many biases in coding
structure of chromosomes. One of the processes affected by the asymmetry is
translocation changing the position of the coding sequence on chromosome in
respect to the orientation on the leading and lagging DNA strand. When analysing
sets of paralogs in 50 genomes, we found that the number of observed genes which
switched their positions on DNA strand is lowest for genomes with the highest
DNA asymmetry. However, the number of orthologs which changed DNA strand
increases with the phylogenetic distance between the compared genomes.
Nevertheless, there is a fraction of coding sequences that stay on the leading
strand in all analysed genomes, whereas there are no sequences that stay always
on the lagging strand. Since sequences diverge very fast after switching the DNA
strand, this bias in mobility of sequences is responsible, in part, for higher
divergence rates among some of coding sequences located on the lagging DNA
strand. In bacteria, most genes are on the leading strand of replication, a phenomenon
attributed to collisions between the DNA and RNA polymerases. In Escherichia
coli, these collisions slow the movement of the replication fork through
actively transcribed genes only if they are coded on the lagging strand. For
genes on both strands, however, these collisions sever nascent transcripts and
interrupt gene expression. Based on these observations, we propose a new theory
to explain strand bias: genes whose expression is important for fitness are
selected to the leading strand because this reduces the duration of these
interruptions. Our theory predicts that multi-gene operons, which are subject to
longer interruptions, should be more strongly selected to the leading strand
than singleton transcripts. We show that this is true even after controlling for
the tendency for essential genes, which are strongly biased to the leading
strand, to occur in operons. Our theory also predicts that other factors that
are associated with strand bias should have stronger effects for genes that are
in operons. We find that expression level and phylogenetic ubiquity are
correlated with strand bias for both essential and non-essential genes, but only
for genes in operons. In this study, the factors driving genome-wide patterns of codon usages in
Lawsonia intracellularis genome are determined. For genes on the chromosome of
the bacterium, it is found that the most important source of variation results
from strand-specific mutational biases. A lesser trend of variation is
attributable to genes that are presumed as horizontally transferred. These
putative alien genes are unusually GC richer than the other genes, whereas
horizontally transferred genes have been observed to be AT rich in bacteria with
medium and relatively low G + C contents. Hydropathy of encoded protein and
expression level are also found to influence codon usage. Therefore, codon usage
in L. intracellularis chromosome is the result of a complex balance among the
different mutational and selectional factors. When analyzing genes in the
largest plasmid, for the first time it is found that the strand-specific
mutational biases are responsible for the primary variation of codon usages in
plasmid. Genes, particularly highly expressed genes of this plasmid, are mainly
located on the leading strands and this supposed to be the effects exerted by
replicational-transcriptional selection. These facts suggest that this plasmid
adopts the similar mechanism of replication as the chromosome in L.
intracellularis. Common characters among the 10 bacteria in whose genomes the
strand-specific mutational biases are the primary source of variation of codon
usage are also investigated. For example, it is found that genes dnaT and fis
that are involved in DNA replication initiation and re-initiation pathways are
absent in all of the 10 bacteria. We studied nucleotide usage biases in 4-fold degenerated sites of all the genes
from leading and lagging strands of 30 bacterial genomes. The level of guanine
in 4-fold degenerated sites (G4f) is significantly lower in genes from lagging
strands than in genes from leading strands, probably because of the faster rates
of guanine oxidation in single-stranded DNA leading to G to T transversions. The
rates of cytosine deamination causing C to T transitions are also higher in
lagging strands. We showed that the level of codons able to form stop-codons by
the way of G to T transversions and C to T transitions is always higher than the
level of codons able to form stop-codons by the way of C to A transversions and
G to A transitions. This circumstance can be an explanation of the lower percent
of ORFs in lagging strands of bacterial replichores than in leading strands. The majority of bacterial genes are located on the leading strand, and the
percentage of such genes has a large variation across different bacteria.
Although some explanations have been proposed, these are at most partial
explanations as they cover only small percentages of the genes and do not even
consider the ones biased toward the lagging strand. We have carried out a
computational study on 725 bacterial genomes, aiming to elucidate other factors
that may have influenced the strand location of genes in a bacterium. Our
analyses suggest that (i) genes of some functional categories such as ribosome
have higher preferences to be on the leading strands; (ii) genes of some
functional categories such as transcription factor have higher preferences on
the lagging strands; (iii) there is a balancing force that tends to keep genes
from all moving to the leading and more efficient strand and (iv) the percentage
of leading-strand genes in an bacterium can be accurately explained based on the
numbers of genes in the functional categories outlined in (i) and (ii), genome
size and gene density, indicating that these numbers implicitly contain the
information about the percentage of genes on the leading versus lagging strand
in a genome. Several mechanisms that increase the rate of mutagenesis across the entire
genome have been identified; however, how the rate of evolution might be
promoted in individual genes is unclear. Most genes in bacteria are encoded on
the leading strand of replication. This presumably avoids the potentially
detrimental head-on collisions that occur between the replication and
transcription machineries when genes are encoded on the lagging strand. Here we
identify the ubiquitous (core) genes in Bacillus subtilis and determine that 17%
of them are on the lagging strand. We find a higher rate of point mutations in
the core genes on the lagging strand compared with those on the leading strand,
with this difference being primarily in the amino-acid-changing (nonsynonymous)
mutations. We determine that, overall, the genes under strong negative selection
against amino-acid-changing mutations tend to be on the leading strand,
co-oriented with replication. In contrast, on the basis of the rate of
convergent mutations, genes under positive selection for amino-acid-changing
mutations are more commonly found on the lagging strand, indicating faster
adaptive evolution in many genes in the head-on orientation. Increased gene
length and gene expression amounts are positively correlated with the rate of
accumulation of nonsynonymous mutations in the head-on genes, suggesting that
the conflict between replication and transcription could be a driving force
behind these mutations. Indeed, using reversion assays, we show that the
difference in the rate of mutagenesis of genes in the two orientations is
transcription dependent. Altogether, our findings indicate that head-on
replication-transcription conflicts are more mutagenic than co-directional
conflicts and that these encounters can significantly increase adaptive
structural variation in the coded proteins. We propose that bacteria, and
potentially other organisms, promote faster evolution of specific genes through
orientation-dependent encounters between DNA replication and transcription. Genomic DNA is used as the template for both replication and transcription,
whose machineries may collide and result in mutagenesis, among other damages.
Because head-on collisions are more deleterious than codirectional collisions,
genes should be preferentially encoded on the leading strand to avoid head-on
collisions, as is observed in most bacterial genomes examined. However, why are
there still lagging strand encoded genes? Paul et al. recently proposed that
these genes take advantage of the increased mutagenesis resulting from head-on
collisions and are thus adaptively encoded on the lagging strand. We show that
the evidence they provided is invalid and that the existence of lagging strand
encoded genes is explainable by a balance between deleterious mutations that
bring genes from the leading to the lagging strand and purifying selection
purging such mutants. Therefore, the adaptive hypothesis is neither
theoretically needed nor empirically supported. |
What hand deformities do patients with Apert syndrome present with? | In patients with Apert syndrome, the hands demonstrate many disturbances of soft tissue and bony structures. These include a short thumb with radial clinodactyly, complex syndactyly with a bony fusion involving the index, long and ring fingers, symphalangism and simple syndactyly of the fourth web space. The soft tissue anomalies involve the intrinsic muscles, the extrinsic tendon insertions and the neurovascular bundles. | A material of 89 cases of upper extremity deformities, among the 3225 cleft
patients born during the period 1950-75, and treated in the Finnish Red Cross
Cleft Centre is presented. About two-thirds of the patients had an isolated
cleft palate--half of the male and nearly all of the female patients. The
percentage of upper extremity deformities appearing with the different types of
the orofacial clefts was, for clefts of the primary palate 2.0: specifically for
cleft lip 0.8, cleft lip--palate 2.6, and cleft lip and palate 3.6; and for
clefts of the secondary palate 3.5: specifically for cleft palate 3.7, submucous
cleft palate 1.6; and for the branchial arch syndrome (lateral cleft) 5.2; the
total average being 2.8 percent. About one-third of the patients were dwarfs,
most of them diastrophic dwarfs. Syndactyly was somewhat more common among cleft
patients, 0.3%, than in the average population. Polydactyly, 0.1% was about as
common as the average. Ectrodactyly was more common among cleft patients, 0.4%,
than either syndactyly or polydactyly that are considered the most common hand
deformities among the general population. The syndactyly cases were more
complicated than the average, among them 4 cases of Apert syndrome were noted.
About three-fourths of the 89 patients had multiple deformities. Surgical correction of syndactyly of the Apert hand should begin by 6 months and
be completed by 3 years of age. As much surgery as possible is carried out at
each sitting. Digit separation should be in order of functional importance. The
first web space is deepened with a four-flap Z-plasty or a dorsal skin flap from
the web and index finger. Syndactyly release using a dorsal flap and zig-zag
technique is used to create the second and fourth web spaces. The complex
long-ring syndactyly often requires a pedicled groin flap for reconstruction and
preservation of growth potential. A five-digit hand can be achieved with
adequate grasp and stable, sensate, well-aligned digits. These children can
attain some degree of independent finger motion and aesthetically acceptable
hands with this approach. Domitly acting, allelic mutations of the fibroblast growth factor receptor 2
(FGFR2) gene have been described in five craniosynostosis syndromes. In Apert
syndrome, characterised by syndactyly of the hands and feet, recurrent mutations
of a serine-proline dipeptide (either Ser252Trp or Pro253Arg) in the linker
between the IgII and IgIII extracellular immunoglobulin-like domains, have been
documented in more than 160 unrelated individuals. We have identified three
novel mutations of this dipeptide, associated with distinct phenotypes. A C-->T
mutation that predicts a Ser252Leu substitution, ascertained in a boy with mild
Crouzon syndrome (craniosynostosis with normal limbs) is also present in three
clinically normal members of his family. A CG-->TT mutation that predicts a
Ser252Phe substitution results in a phenotype consistent with Apert syndrome.
Finally, a CGC-->TCT mutation that predicts a double amino acid substitution
(Ser252Phe and Pro253Ser) causes a Pfeiffer syndrome variant with mild
craniosynostosis, broad thumbs and big toes, fixed extension of several digits,
and only minimal cutaneous syndactyly. The observation that the Ser252Phe
mutation causes Apert syndrome, whereas the other single or double substitutions
are associated with milder or normal phenotypes, highlights the exquisitely
specific molecular pathogenesis of the limb and craniofacial abnormalities
associated with Apert syndrome. Ser252Phe is the first noncanonical mutation to
be identified in this disorder, its rarity being explained by the requirement
for two residues of the serine codon to be mutated. The description of
independent, complex nucleotide substitutions involving identical nucleotides is
unprecedented, and we speculate that this may result from functional selection
of FGFR mutations in sperm. Apert syndrome, characterised by craniosynostosis, craniofacial anomalies, and
symmetrical syndactyly of the digits (cutaneous and bony fusion), has been
associated with two canonical mutations in the FGFR2 gene (S252W, P253R) in the
great majority of cases. Since these two alterations have been observed
exclusively among these patients, it has been suggested that the S252W and P253R
changes may play an important role in the occurrence of syndactyly. In order to
verify whether the mutations S252W and P253R could also cause a milder
phenotype, without involvement of the limbs, we have screened 22 patients with
clinical characteristics compatible with Crouzon or Pfeiffer syndrome for these
two particular changes. Surprisingly, we identified a Pfeiffer-like patient with
the mutation S252W, and therefore we have shown for the first time the
occurrence of one of the canonical Apert mutations without severe abnormalities
of the upper and lower extremities. Children born with Apert acrocephalosyndactyly pose great challenges to the
pediatric hand surgeon. Reconstructive dilemmas consist of shortened, deviated
phalanges and extensive skin deficits following syndactyly release. We present a
10-year review of patients with Apert acrocephalosyndactyly who were treated
with a simplified surgical approach. Between 1986 and 1996, 10 patients with
Apert syndrome underwent reconstructive surgery of their hands. The overall
strategy involved early bilateral separation of syndactylous border digits at 1
year of age, followed by sequential unilateral middle syndactyly mass separation
with thumb osteotomy and bone grafting as needed. In these 10 patients, a total
of 53 web spaces were released, 49 of which involved osteotomies for complex
syndactyly. Only local flaps and full-thickness skin grafts from the groin were
used in all cases to achieve soft-tissue coverage. To date, seven of the 53 web
spaces have needed revision (revision rate, 13 percent). Eleven thumb
osteotomies (nine opening wedge and two closing wedge) were performed. Bone
grafts from the proximal ulna or from other digits were used in all cases. To
date, none of these thumb osteotomies have needed revision. This early,
simplified approach to the complex hand anomalies of Apert acrocephalosyndactyly
has been successful in achieving low revision rates and excellent functional
outcomes as measured by gross grasp and pinch and by patient and parent
satisfaction. In patients with Apert syndrome, the hands demonstrate many disturbances of soft
tissue and bony structures. These include a short thumb with radial
clinodactyly, complex syndactyly with a bony fusion involving the index, long
and ring fingers, symphalangism and simple syndactyly of the fourth web space.
The soft tissue anomalies involve the intrinsic muscles, the extrinsic tendon
insertions and the neurovascular bundles. We have reviewed 52 patients who
underwent surgical reconstruction of their hands. The aim of this study is to
propose a better surgical management in the light of recent publications and to
improve our understanding of the syndrome, attempting to reduce the number of
procedures and to select the best possible procedures for each patient. PURPOSE: To demonstrate the utility of computed tomography angiographic planning
of a single-stage, complete release of syndactyly in Apert syndrome.
METHODS: Computed tomography angiograms were performed as a preoperative
planning tool in 6 patients. Five came to surgery. All had a single-stage
operation for complete release of their syndactyly.
RESULTS: Five patients, ranging from Upton type 1 to type 3 Apert hand
deformities, have had preoperative computed tomography angiography that
delineated the vascular anatomy. This allowed planning and execution of a
single-stage syndactyly release in all patients. The preoperative imaging
identified noteworthy abnormalities in vascular anatomy that were incorporated
into surgical planning.
CONCLUSIONS: The protocol presented allows preoperative planning and
single-stage operation for complete release of syndactyly in patients with Apert
syndrome. |
Is c-myc subject to regulation by the circadian clock? | Yes, the expression of c-myc is regulated by the circadian clock protein Per2. | Period (Per) genes are key circadian rhythm regulators in mammals. Expression of
mouse Per (mPer) genes has a diurnal pattern in the suprachiasmatic nucleus and
in peripheral tissues. Genetic ablation mPER1 and mPER2 function results in a
complete loss of circadian rhythm control based on wheel-running activity in
mice. In addition, these animals also display apparent premature aging and a
significant increase in neoplastic and hyperplastic phenotypes. When challenged
by gamma radiation, mPer2-deficient mice respond by rapid hair graying, are
deficient in p53-mediated apoptosis in thymocytes, and have robust tumor
occurrences. Studies have demonstrated that the circadian clock function is very
important for cell cycle, DNA damage response, and tumor suppression in vivo.
The temporal expression of genes involved in cell cycle regulation and tumor
suppression, such as c-Myc, Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is
deregulated in mPer2 mutant mice. Genetic studies have demonstrated that many
key regulators of cell cycle and growth control are also important circadian
clock regulators, confirming the critical role of circadian function in
organismal homeostasis. The Period (Per) genes are key circadian rhythm regulators in mammals.
Expression of the mouse Per (mPer) genes have diurnal pattern in the
suprachiamstic nuclei and in peripheral tissues. Genetic ablation mPER1 and
mPER2 function results in a complete loss of circadian rhythm control based on
wheel running activity in mice. In addition, these animals also display apparent
premature aging and significant increase in neoplastic and hyperplastic
phenotypes. When challenged by gamma-radiation, mPer2 deficient mice response by
rapid hair graying, are deficient in p53-mediated apoptosis in thymocytes and
have robust tumor occurrences. Our studies have demonstrated that the circadian
clock function is very important for cell cycle, DNA damage response and tumor
suppression in vivo. Temporal expression of genes involved in cell cycle
regulation and tumor suppression, such as c-Myc, Cyclin D1, Cyclin A, Mdm-2 and
Gadd45alpha is deregulated in mPer2 mutant mice. In addition, genetic studies
have demonstrated that many key regulators of cell cycle and growth control are
also important circadian clock regulators confirming the critical role of
circadian function in organismal homeostasis. Recently studies of human breast
and endometrial cancers revealed that the loss and deregulation of PERIOD
proteins is common in the tumor cells. The Period2 gene, an indispensable component of the circadian clock, not only
modulates circadian oscillations, but also regulates organic function. We
examined whether overexpression of the mouse Period2 gene (mPer2) in tumor cells
influences cell growth and induces apoptosis. Overexpression of PERIOD2 in the
mouse Lewis lung carcinoma cell line (LLC) and mammary carcinoma cell line
(EMT6) results in reduced cellular proliferation and rapid apoptosis, but not in
NIH 3T3 cells. Overexpressed mPER2 also altered the expression of
apoptosis-related genes. The mRNA and protein levels of c-Myc, Bcl-X(L) and
Bcl-2 were downregulated, whereas the expression of p53 and bax was upregulated
in mPER2-overexpressing LLC cells compared with control cells transferred with
empty plasmid. Our results suggest that the circadian gene mPeriod2 may play an
important role in tumor suppression by inducing apoptotic cell death, which is
attributable to enhanced pro-apoptotis signaling and attenuated anti-apoptosis
processes. Colorectal cancer risk is increased in shift workers with presumed circadian
disruption. Intestinal epithelial cell proliferation is gated throughout each
day by the circadian clock. Period 2 (Per2) is a key circadian clock gene. Per2
mutant (Per2(m/m)) mice show an increase in lymphomas and deregulated expression
of cyclin D and c-Myc genes that are key to proliferation control. We asked
whether Per2 clock gene inactivation would accelerate intestinal and colonic
tumorigenesis. The effects of PER2 on cell proliferation and beta-catenin were
studied in colon cancer cell lines by its down-regulation following RNA
interference. The effects of Per2 inactivation in vivo on beta-catenin and on
intestinal and colonic polyp formation were studied in mice with Per2 mutation
alone and in combination with an Apc mutation using polyp-prone Apc(Min/+) mice.
Down-regulation of PER2 in colon cell lines (HCT116 and SW480) increases
beta-catenin, cyclin D, and cell proliferation. Down-regulation of beta-catenin
along with Per2 blocks the increase in cyclin D and cell proliferation.
Per2(m/m) mice develop colonic polyps and show an increase in small intestinal
mucosa beta-catenin and cyclin D protein levels compared with wild-type mice.
Apc(Min/+)Per2(m/m) mice develop twice the number of small intestinal and
colonic polyps, with more severe anemia and splenomegaly, compared with
Apc(Min/+) mice. These data suggest that Per2 gene product suppresses
tumorigenesis in the small intestine and colon by down-regulation of
beta-catenin and beta-catenin target genes, and this circadian core clock gene
may represent a novel target for colorectal cancer prevention and control. Disruption of circadian rhythms, daily oscillations in biological processes that
are regulated by an endogenous clock, has been linked to tumorigenesis. Normal
and maligt tissues often show asynchronies in cell proliferation and
metabolic rhythms. Cancer chronotherapy takes biological time into account to
improve the therapy. However, alterations of the circadian clock machinery genes
have rarely been reported in human cancer. Herein, we show that the BMAL1 gene,
a core component of the circadian clock, is transcriptionally silenced by
promoter CpG island hypermethylation in hematologic maligcies, such as
diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We
also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma
cells causes growth inhibition in colony assays and nude mice, whereas BMAL1
depletion by RNA interference in unmethylated cells enhances tumor growth. We
also show that BMAL1 epigenetic inactivation impairs the characteristic
circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in
association with a loss of BMAL1 occupancy in their respective promoters.
Furthermore, the DNA hypermethylation-associated loss of BMAL1 also prevents the
recruitment of its natural partner, the CLOCK protein, to their common targets,
further enhancing the perturbed circadian rhythm of the maligt cells. These
findings suggest that BMAL1 epigenetic inactivation contributes to the
development of hematologic maligcies by disrupting the cellular circadian
clock. Per2 regulates other molecular and biochemical processes beyond their
established role in the regulation of the mammalian circadian clock, herein we
investigated the growth inhibiting potential of Per2 in human K562 leukemia
cells and the underlying mechanisms. The results showed that over-expression of
Per2 induced not only cell cycle arrest at G2/M phase but also an increase in
apoptosis, which was confirmed by characteristic morphological changes, FCM and
evident DNA fragmentation. Further experiments confirmed both up-regulation of
P53 and down-regulation of CylinB1and C-myc. On the other hand, while P53 was
found to be down-regulated. CylinB1 and C-myc were up-regulated. after Per2
knockdown. In leukemia mice, Per2 transfection was shown to suppress cellular
proliferation and accelerate apoptosis of K562 cells. Moreover, fewer leukemia
cells were found to have infiltrated into the livers and spleens of the mice
from the Per2 transfected group as compared with those from the control group.
In summary, Per2 displayed a significant anti-tumor effect through cell cycle
arrest and apoptosis induction in K562 cells. These data further support the
emerging role of the circadian clock in critical aspects of cancer development
and thorough research is underway on the mechanism of Per2 in the leukemia. Circadian rhythms are endogenous and self-sustained oscillations of multiple
biological processes with approximately 24-h rhythmicity. Circadian genes and
their protein products constitute the molecular components of the circadian
oscillator that form positive/negative feedback loops and generate circadian
rhythms. The circadian regulation extends from core clock genes to various
clock-controlled genes that include various cell cycle genes. Aberrant
expression of circadian clock genes, therefore, may lead to genomic instability
and accelerated cellular proliferation potentially promoting carcinogenesis. The
current study encompasses the investigation of simultaneous expression of four
circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled
cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of
serum melatonin levels in peripheral blood samples of 37 CLL (chronic
lymphocytic leukemia) patients and equal number of age- and sex-matched healthy
controls in order to indicate association between deregulated circadian clock
and manifestation of CLL. Results showed significantly down-regulated expression
of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc
and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When
expression of these genes was compared between shift-workers and
non-shift-workers within the CLL group, the expression was found more aberrant
in shift-workers as compared to non-shift-workers. However, this difference was
found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum
melatonin levels were found significantly low (P < 0.0001) in CLL subjects as
compared to healthy controls whereas melatonin levels were found still lower in
shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our
results suggest that aberrant expression of circadian clock genes can lead to
aberrant expression of their downstream targets that are involved in cell
proliferation and apoptosis and hence may result in manifestation of CLL.
Moreover, shift-work and low melatonin levels may also contribute in etiology of
CLL by further perturbing of circadian clock. |
What disease is Velcade (bortezomib) mainly used for? | Velcade (bortezomid), a proteasome inhibitor drug indicated for multiple myeloma (MM) treatment. Velcade is also approved for the treatment of patients with mantle cell lymphoma. | Proteasome inhibitors, a novel class of chemotherapeutic agents, enhance the
antitumor efficacy of anthracyclines in vitro and in vivo. We therefore sought
to determine the maximum tolerated dose (MTD) and dose-limiting toxicities of
bortezomib and pegylated liposomal doxorubicin (PegLD). Bortezomib was given on
days 1, 4, 8, and 11 from 0.90 to 1.50 mg/m2 and PegLD on day 4 at 30 mg/m2 to
42 patients with advanced hematologic maligcies. Grade 3 or 4 toxicities in
at least 10% of patients included thrombocytopenia, lymphopenia, neutropenia,
fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea.
The MTD based on cycle 1 was 1.50 and 30 mg/m2 of bortezomib and PegLD,
respectively. However, due to frequent dose reductions and delays at this level,
1.30 and 30 mg/m2 are recommended for further study. Pharmacokinetic and
pharmacodynamic studies did not find significant drug interactions between these
agents. Antitumor activity was seen against multiple myeloma, with 8 of 22
evaluable patients having a complete response (CR) or near-CR, including several
with anthracycline-refractory disease, and another 8 having partial responses
(PRs). One patient with relapsed/refractory T-cell non-Hodgkin lymphoma (NHL)
achieved a CR, whereas 2 patients each with acute myeloid leukemia and B-cell
NHL had PRs. Bortezomib/PegLD was safely administered in this study with
promising antitumor activity, supporting further testing of this regimen. BACKGROUND: Bortezomib, a first-in-class proteasome inhibitor, has shown
clinical activity in relapsed, refractory multiple myeloma in a pivotal Phase II
trial, SUMMIT.
METHODS: Patients received bortezomib 1.3 mg/m(2) on Days 1, 4, 8, and 11
followed by a 10-day rest period for up to 8 cycles. Dexamethasone 20 mg on the
day of and the day after bortezomib was permitted for suboptimal response.
Extended treatment beyond 8 cycles was offered to patients whose physicians felt
they would benefit from additional therapy. Follow-up was conducted in all
patients for a median of 23 months, an additional 13 months from the original
report.
RESULTS: Of 202 patients enrolled in SUMMIT, 193 were evaluable for response.
Seven (4%) patients achieved a complete response, 12 (6%) achieved a nearly
complete response, 34 (18%) achieved a partial response, and 14 (7%) had a
minimal response while on bortezomib. The updated median duration of response to
bortezomib alone was 12.7 months. The median overall time to progression for all
SUMMIT patients was 7 months. For responding patients, the median time to
progression was 13.9 months, whereas for those with progressive disease (PD) or
who were not evaluable, the median time to progression was 1.3 months. The
median overall survival (OS) for all SUMMIT patients was 17.0 months. Whereas
the median OS for patients with PD or who were not evaluable was 8 months, the
median OS for responding patients was not reached at 23 months of follow-up.
CONCLUSIONS: These data demonstrate that treatment with bortezomib results in
meaningful long-term benefit for patients with relapsed and refractory myeloma. The proteasome plays a pivotal role in the turnover of regulatory transduction
proteins induced by activated cell membrane growth factor receptors. The
epidermal growth factor receptor (EGFR) pathway is crucial in the development
and progression of human epithelial cancers. Proteasome inhibition may sensitize
human cancer cell lines to EGFR inhibitors. We investigated the growth
inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in
combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab
in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent
growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42
nM). A significant synergistic growth inhibitory effect was observed with the
combination of bortezomib and each EGFR inhibitor in all cell lines (combination
index, CI, range 0.10-0.55), which was accompanied by a significant induction in
apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib.
In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR
inhibitor treatment induced a more effective inhibition of EGFR-activated
down-stream signals, including a marked suppression in activated, phosphorylated
Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt
protected A549 cells by cell growth inhibition and apoptosis following treatment
with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and
EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity
in different human cancer cells which possess a functional EGFR-dependent
autocrine growth pathway through to a more efficient and sustained inhibition of
Akt. Relapsed/refractory myeloma has a poor outcome because of multi-drug resistance,
patient low-performance status and toxicity of conventional chemotherapy. To
improve results, standard chemotherapeutics and drugs targeting the
microenvironment are applied at the same time. Bortezomib, by inhibiting
proteasome function, may enhance chemosensitivity to other drugs and overcome
drug-resistance. Notably, doxorubicin and bortezomib may reciprocally increase
their efficacy. Thus, to improve outcome whilst minimizing therapy-related
toxicity, liposomal doxorubicin was added to a bortezomib-based combination.
From January 2004, relapsed/refractory myeloma patients referred to our
Institution received bortezomib 1.0 mg/m(2) i.v. twice weekly for 2 weeks in a
28-d cycle for up to six cycles, oral dexamethasone 24 mg with the standard
scheduling and thalidomide 100 mg continuously (VTD). From January 2005,
liposomal doxorubicin, 50 mg/m(2) (30 mg/m(2) for patients older than 75 years),
was added on day 4 of each cycle [VTD plus Myocet (MyVTD)]. In total, 70
patients were treated: 28 received VTD and 42 MyVTD. Baseline demographic and
clinical characteristics were similar between the two groups. Toxicity was
manageable although more pronounced with MyVTD. The overall response rate (81%
vs. 50%, P = 0.009), time to progression (19 vs. 11 months, P = 0.01) and
progression-free survival (15 vs. 8 months, P = 0.001) were significantly higher
with MyVTD regimen, suggesting an improved quality of response. The anticancer potency of green tea and its individual components is being
intensely investigated, and some cancer patients already self-medicate with this
"miracle herb" in hopes of augmenting the anticancer outcome of their
chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for
multiple myeloma. Here, we investigated whether the combination of these
compounds would yield increased antitumor efficacy in multiple myeloma and
glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that
various green tea constituents, in particular (-)-epigallocatechin gallate
(EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively
prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced
antagonistic function of EGCG was evident only with boronic acid-based
proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non-boronic
acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted
with BZM and blocked its proteasome inhibitory function; as a consequence, BZM
could not trigger endoplasmic reticulum stress or caspase-7 activation, and did
not induce tumor cell death. Taken together, our results indicate that green tea
polyphenols may have the potential to negate the therapeutic efficacy of BZM and
suggest that consumption of green tea products may be contraindicated during
cancer therapy with BZM. OBJECTIVE: To evaluate the effects and safety of the regimen of bortezomib
combined with dexamethasone (VD) in the treatment of primary systemic (AL)
amyloidosis.
METHODS: Five newly diagnosed AL amyloidosis patients confirmed by renal biopsy
with a median of 3 organs involved (3 to 5 organs) were treated by VD regimen
for 3 (1-4) cycles.
RESULTS: Among 3 evaluable patients, 1 was in stable condition and 2 had
hematologic response (partial remission and complete remission) and organ
function improvement. Hematologic responses were rapid (median 1.5 cycles) and
median time to organ response was 2 cycles. Three cases were survived and the
periods of follow up were 5, 4 and 4 months respectively. The other 2 died 2 and
14 months after diagnosis. The side effects were asthenia, diarrhea,
constipation, edema aggravation and fever, all of which were in I grade. No
treatment associating death was found.
CONCLUSION: VD regimen might be an efficient, rapid effective and safe regimen
in the treatment of AL amyloidosis. PURPOSE: We studied the efficacy and safety of bortezomib (BOR) for treatment of
multiple myeloma in comparison with thalidomide (THAL) by reference to adverse
events, and searched for laboratory markers that could be used for
prognostication of patients.
METHODS: Biochemical data of patients receiving BOR and THAL for treatment of
multiple myeloma at the Japanese Red Cross Narita Hospital were investigated
retrospectively, after obtaining Institutional Review Board approval. Judgment
of curative effects complied with the effects criteria of the International
Myeloma Working Group (IMWG).
RESULTS: BOR showed a higher rate of effectiveness than THAL for refractory
multiple myeloma, and its effects were rapid. BOR treatment prolonged the
survival time of THAL-resistant patients. The efficacy of BOR was unrelated to
patient age, the number of previous therapeutic regimens, or the disease period.
After medication with BOR, patients in whom it had been effective tended to show
an increase of the serum alkaline phosphatase (ALP) level. Thrombocytopenia
(86.2%) and leucopenia (69.0%) were observed at high frequencies, but no
previously unreported adverse events or fatalities were associated with BOR
therapy.
CONCLUSION: It is suggested that BOR has therapeutic efficacy for multiple
myeloma as a first-line medical treatment and/or for patients with THAL
resistance, and can improve prognosis and survival. Since serum ALP elevation
was observed in many patients for whom BOR was effective, this may be a
predictor of BOR efficacy. Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S
proteasome, is remarkably effective against multiple myeloma (MM) but not
against solid tumors. Dose-limiting adverse effects from "on target" inhibition
of the proteasome in normal cells and tissues appear to be a key obstacle.
Achieving efficacy against solid tumors therefore is likely to require making
the inhibitor more selective for tumor tissue over normal tissues. The simplest
strategy that might provide such tissue specificity would be to employ a tumor
specific protease to release an inhibitor from a larger, noninhibitory
structure. However, such release would necessarily generate an inhibitor with a
free N-terminal amino group, raising a key question: Can short peptide boronic
acids with N-terminal amino groups have the requisite properties to serve as
warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest
plausible candidates for the task, can indeed be sufficiently potent,
cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to
serve in this capacity. OBJECTIVES: Single-size vials of drugs may be a source of waste and increase in
treatment costs. Bortezomib, indicated for multiple myeloma (MM) treatment, is
available in 3.5-mg vials, a quantity higher than the average dose commonly
prescribed. This analysis aimed to demonstrate, through real-world data, which
would be the optimal vial presentation for bortezomib in Brazil and quantify the
reduction in medication waste related to this option.
METHODS: From November 2007 to October 2009 all patients with MM treated with
bortezomib were identified via the Evidências database. Analysis of prescribed,
dispensed, and wasted doses, their costs and projections of the ideal vial size
were performed.
RESULTS: Thirty-five patients (mean body surface area of 1.73 m(2)) received 509
infusions in 131 cycles of treatment (average of 3.77 cycles per patient). The
average dose prescribed was 2.1 mg per infusion (95% confidence interval [CI]
1.97-2.26) with average waste of 39.5% of the vial content (95% CI 35.35-43.76).
The mean waste per patient per day was 1.38 mg (95% CI 1.24-1.52). If a 3-mg
vial were available, the average drug waste per patient per day would be 0.88 mg
(95% CI 0.74-1.03) or 36.2% less. With a 2.5-mg vial the waste would be 1.05 mg
(95% CI 0.81-1.29) or 23.9% less. If two presentations were available (2.5 mg
and 0.5 mg), the waste would be 0.52 mg (95% CI 0.4-0.63) or 62.5% less.
Considering the price of the different vials to be proportional to the original
3.5-mg vial, the cost would be also reduced by the same rates described above.
CONCLUSIONS: A simple adjustment in vial size may reduce the waste of bortezomib
by 36% to 62% and can also reduce the cost of treatment. The ascertainment of serum free light chain (sFLC) levels has been shown to be
valuable in screening for the presence of plasma cell dyscrasia as well as for
baseline prognosis in newly diagnosed patients. For patients with amyloidosis
and those with oligo-secretory or non-secretory multiple myeloma (MM), serial
measurement of sFLC has also been shown to be valuable in monitoring disease
status. However, in patients with a measureable, intact monoclonal protein by
immunofixation (M protein), the serial measurement of sFLC remains undefined and
is currently not recommended in professional guidelines. Herein, we provide data
comparing sFLC with M protein as biomarkers of response in newly diagnosed
patients with MM undergoing induction therapy with the novel agents thalidomide,
lenalidomide and/or bortezomib. We show that although M protein appears to
outperform sFLC comparatively over the course of induction therapy, the addition
of FLC to M protein further informs the characterization of residual disease
status post-induction. Moreover, sFLC at the time of stem cell mobilization
appears to hold prognostic power for survival endpoints following high-dose
chemotherapy/autologous stem cell transplant (HDC/SCT). These findings suggest
potentially novel roles for sFLC in patients with MM with an intact M protein
receiving novel agent-based induction strategies followed by HDC/SCT. BACKGROUND AND OBJECTIVE: Bortezomib, an antineoplastic agent with proteasome
inhibitory activity, is extensively metabolized by the hepatic microsomal
cytochrome P450 (CYP) enzymes CYP3A4 and CYP2C19. Drugs that affect these
enzymes may therefore have an impact on the pharmacological profile of
bortezomib. This study evaluated the effects of co-administration of a potent
CYP3A4 inducer (rifampicin [rifampin]) and a weak CYP3A4 inducer (dexamethasone)
on the pharmacokinetic, pharmacodynamic and safety profiles of bortezomib.
PATIENTS AND METHODS: Patients aged ≥18 years with relapsed or refractory
multiple myeloma or non-Hodgkin's lymphoma received intravenous bortezomib
1.3 mg/m2, administered on days 1, 4, 8 and 11 of a 21-day cycle, for 3 cycles.
In stage 1, patients were randomized (1 : 1) to receive bortezomib alone or in
combination with oral rifampicin 600 mg once daily on days 4-10 during cycle 3
only. If the mean area under the plasma concentration-time curve (AUC) of
bortezomib was reduced by ≥30% during rifampicin co-administration, then stage 2
was initiated, in which patients received bortezomib with dexamethasone 40 mg
once daily on days 1-4 and days 9-12 during cycle 3 only. Blood samples were
collected on days 11 through 14 of cycles 2 and 3 before and after bortezomib
administration, at prespecified time points, for pharmacokinetic and
pharmacodynamic (proteasome inhibition) assessments.
RESULTS: Twelve patients in the bortezomib-alone arm, six patients in the
bortezomib plus rifampicin arm and seven patients in the bortezomib plus
dexamethasone arm were included in the pharmacokinetics-evaluable set.
Rifampicin reduced the mean AUC from 0 to 72 hours (AUC(72h)) of bortezomib by
approximately 45% (223 ng · h/mL in cycle 2 vs 123 ng · h/mL in cycle 3), while
dexamethasone had no effect (mean AUC(72h): 179 ng · h/mL in cycle 2 vs
170 ng · h/mL in cycle 3). Proteasome inhibition parameters in peripheral blood
were unaffected by rifampicin or dexamethasone. Safety profiles were similar
across the treatment arms and consistent with previous experience of bortezomib.
CONCLUSIONS: In patients with multiple myeloma or non-Hodgkin's lymphoma,
co-administration of rifampicin decreased the exposure to bortezomib but did not
affect the proteasome inhibition or safety profiles; co-administration of
dexamethasone did not affect the exposure to bortezomib, proteasome inhibition
or safety profiles. Concomitant administration of bortezomib with strong CYP3A4
inducers such as rifampicin is not recommended, as it may result in a reduction
of the clinical effect, whereas concomitant administration of weak CYP3A4
inducers such as dexamethasone does not affect the pharmacological profile of
bortezomib. BACKGROUND: Thalidomide was approved in Japan for multiple myeloma treatment in
October 2008. A program called the Thalidomide Education and Risk Management
System (TERMS®) was established to help ensure that every effort is made to use
the drug safely.
PURPOSE: We report the use of thalidomide to treat multiple myeloma, and
describe problems arising in the Thaled® outpatient department.
PATIENTS AND METHODS: Multiple myeloma patients treated with thalidomide at
Hitachi General Hospital.
INTERVENTION: Monitoring of the efficacy and safety of thalidomide, and a
questionnaire survey conducted at the Thaled® outpatient department.
RESULTS: The thalidomide response rate was 41. 7%. In 5 cases, all patients
received steroids along with thalidomide. After auto-PBSCT, 1 of 2 cases
demonstrated a good response (PR 1). After treatment with bortezomib, 1 of 2
cases demonstrated a good response (MR 1). After auto-PBSCT and treatment with
bortezomib, 1 of 4 cases demonstrated a good response (PR 1). In a case
demonstrating hematotoxicity Grade 3 (in addition to neutropenia),
administration was discontinued. Regarding problems in the Thaled® outpatient
department, the medical staff indicated that TERMS® is a very complicated
program, while the patients requested prolongation of the prescription days and
reduction of the economic burden of medication costs.
CONCLUSION: Thalidomide showed some success in treating multiple myeloma either
after auto-PBSCT or following treatment with bortezomib. In the case
demonstrating hematotoxicity Grade 3 (in addition to neutropenia), grave
complications could have very easily developed, thus underscoring the importance
of careful monitoring. Based on a questionnaire survey conducted in the Thaled®
outpatient department, the medical staff made comments and patients raised
issues that should be examined in the future. Bortezomib is well-known for inducing cell death in cancer cells, specifically
through the mechanism of proteasome inhibition. Thiostrepton, a thiazole
antibiotic, has also been described for its proteasome inhibitory action,
although differing slightly to bortezomib in the proteasomal site to which it is
active. Previously we had shown the synergic effect of bortezomib and
thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations
of both proteasome inhibitors resulted in synergic increase in cell death when
combined as a treatment. Here, we administered such a combination to MDA-MB-231
xenograft tumors in vivo, and found that the effect of complementary proteasome
inhibitors reduced tumor growth rates more efficiently than compared with when
administered alone. Increased induction of apoptotic activity in tumors was
found be associated with the growth inhibitory activity of combination
treatment. Further examination additionally revealed that combination-treated
tumors exhibited reduced proteasome activity, compared with non-treated and
single drug-treated tumors. These data suggest that this drug combination may be
useful as a therapy for solid tumors. PURPOSE: Bortezomib, a proteasome inhibitor drug very effective against multiple
myeloma, may induce the so-called bortezomib-induced peripheral neuropathy
(BIPN), hardly manageable with common analgesic drugs. This study assessed the
effectiveness of controlled-release (CR) oral oxycodone in controlling pain and
its interference on daily functions of patients with hematologic maligcies
affected by BIPN.
METHODS: Forty-six patients (median age, 62 years) affected by myeloma and
lymphoma, complaining of BIPN-related pain of moderate-to-severe intensity and
unresponsive to previous analgesic treatments, were treated with CR oxycodone.
The intensity of continuous and brief pain (BP) along with interference of pain
with the common daily dimensions of feeling and function were evaluated by using
an 11-point numerical rating scale (NRS); a global patient evaluation of
efficacy was also performed.
RESULTS: The daily average dose of CR oxycodone administered was 28.46 mg
(range, 20-80 mg). The pain intensity decreased from a mean NRS value of 7.6 at
baseline to 1.3 on day 14. The frequency of BP was reduced from 61 to 47% of
patients and its intensity from 7.4 to 3.1 NRS score. A similar trend to
decreasing values was observed for all the daily life functions. Slight- or
mild-intensity side effects were observed in 23 patients (51%). At the end of
the study, 75% of patients found the treatment effective or very effective.
CONCLUSION: CR oxycodone for relief of BIPN-related pain was effective and well
tolerated. The pain control significantly improved also the quality of the daily
life functions, which are usually compromised in these suffering patients. |
Which genes are thought to be involved in medulloblastoma development? | Medulloblastomas are the most frequent malignant brain tumors affecting children. Disease development has been suggested to be associated with a significant number of genes, such as PTCH1, SUFU, PTEN, CREBBP, PTEN, MYT1L, NFIA, NFIB, TEAD1, TGIF2, IGF2, PCDH10, BMI1, MYC, OTX2, RASSF1A, HIC1, and CASP8. | Epigenetic inactivation of the RASSF1A tumor suppressor gene (TSG) at chromosome
3p21.3 was examined in medulloblastoma, the most common maligt brain tumor of
childhood. Seventy-nine % (27 of 34) of primary tumors and 100% (8 of 8) of
medulloblastoma cell lines displayed extensive tumor-specific DNA
hypermethylation across the RASSF1A promoter-associated CpG island.
Hypermethylation was associated with epigenetic silencing of RASSF1A
transcription in medulloblastoma cell lines, and RASSF1A expression in these
lines was restored after treatment with the DNA-methyltransferase inhibitor
5-aza-2'-deoxycytidine. No evidence was found of RASSF1A inactivation by genetic
mechanisms (gene mutation or deletion) in either cases with no evidence of
RASSF1A hypermethylation or paired normal/tumor cases and cell lines with
evidence of total RASSF1A CpG island hypermethylation. Epigenetic inactivation
by biallelic hypermethylation therefore represents the primary mechanism of
RASSF1A gene inactivation in medulloblastoma. Furthermore, RASSF1A
hypermethylation is a frequent event in medulloblastoma tumorigenesis detectable
in adult (5 of 7) and pediatric patients (22 of 27) and in all histological
variants and age and sex groupings. Importantly, these data demonstrate that
comprehensive analysis of the genome and epigenome will be required for
identification of the key tumor suppressor genes involved in medulloblastoma
development. Medulloblastoma arises in the cerebellum and is the most common maligt brain
tumour of childhood, however its molecular basis is not well understood. To
assess the role of aberrant epigenetic events in medulloblastoma and identify
critical genes in its development, we profiled the promoter methylation status
of 11 candidate tumour-suppressor genes (TSGs; p14(ARF), p15(INK4b), p16(INK4a),
CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma
cell lines, primary tumours and the normal cerebellum. Gene-specific TSG
methylation was a significant feature of both medulloblastomas and the
cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in
medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus
0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a
subset of primary tumours (17/44 and 14/39) occurred against a consistent
background of partial methylation in the normal cerebellum. These data therefore
indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are
tumour-specific events in medulloblastoma. Moreover, methylation of these genes
in medulloblastoma cell lines was associated with their epigenetic
transcriptional silencing and methylation-dependent re-expression following
treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. The
remaining genes studied showed either low frequency methylation (p14(ARF),
p16(INK4a), RIZ1; <7% of cases), no evidence of methylation (p15(INK4b), TIMP3,
TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum
(EDNRB), suggesting that their hypermethylation does not play a major role in
medulloblastoma. Our data demonstrate that tumour-specific hypermethylation
affects only a subset of genes, and does not support the existence of a
concordant methylation phenotype in this disease. We conclude that epigenetic
TSG inactivation is a significant feature of medulloblastoma, and identify
RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis.
Furthermore, methylation observed in the normal cerebellum emphasises the
requirement for appropriate control tissues when assessing the
tumour-specificity of TSG hypermethylation. Inappropriate Hedgehog (Hh) signaling underlies development of a subset of
medulloblastomas, and tumors with elevated HH signaling activity express the
stem cell self-renewal gene BMI1. To test whether Bmi1 is required for Hh-driven
medulloblastoma development, we varied Bmi1 gene dosage in transgenic mice
expressing an oncogenic Hh effector, SmoA1, driven by a glial fibrillary acidic
protein (GFAP) promoter. Whereas 100% of SmoA1; Bmi1(+/+) or SmoA1;Bmi1(+/-)
mice examined between postnatal (P) days 14 and 26 had typical medulloblastomas
(N = 29), tumors were not detected in any of the SmoA1;Bmi1(-/-) animals
examined (N = 6). Instead, small ectopic collections of cells were present in
the region of greatest tumor load in SmoA1 animals, suggesting that
medulloblastomas were initiated but failed to undergo expansion into frank
tumors. Cells within these Bmi1(-/-) lesions expressed SmoA1 but were largely
nonproliferative, in contrast to cells in Bmi1(+/+) tumors (6.2% vs 81.9%
PCNA-positive, respectively). Ectopic cells were negative for the progenitor
marker nestin, strongly GFAP-positive, and highly apoptotic, relative to
Bmi1(+/+) tumor cells (29.6% vs 6.3% TUNEL-positive). The alterations in
proliferation and apoptosis in SmoA1;Bmi1(-/-) ectopic cells are associated with
reduced levels of Cyclin D1 and elevated expression of cyclin-dependent kinase
inhibitor p19(Arf), two inversely regulated downstream targets of Bmi1. These
data provide the first demonstration that Bmi1 is required for spontaneous de
novo development of a solid tumor arising in the brain, suggest a crucial role
for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma
expansion, and implicate Bmi1 as a key factor required for Hh pathway-driven
tumorigenesis. INTRODUCTION: Medulloblastoma is the most frequent type of embryonal tumor in
the pediatric population, accounting for 20-25% of all brain tumors in children.
Recently, the suspected contribution of the Polycomb group (PcG) genes in
medulloblastoma development was described. PcG genes play an important role in
developmental processes; they are also involved in the self-renewal of
hematopoietic and neural stem cells as well as in maligt transformation.
PURPOSE: In this study, we evaluated the expression of BMI1and PCGF2, members of
family of PcG genes, and their potential target, MYC oncogene, and analyzed
their association with demographic and clinical data.
MATERIALS AND METHODS: Thirty-one children (18 males and 13 females, aged from
0.4 to 17 years) with medulloblastoma were included in this study. The gene's
expression level was measured by quantitative real-time PCR, obtained using the
two-color multiplexing technique.
RESULTS: We found that the higher expression levels of BMI1 and PCGF2 genes were
associated with significantly decreased patient survival (p = 0.02 and
p = 0.012, respectively). Significant differences between gender were found,
with a higher expression level of the PCGF2 gene observed among females
(p = 0.02).
CONCLUSION: Our analysis showed correlation between BMI1 and PCGF2 gene's
expression and survival in children with medulloblastoma. BACKGROUND: Medulloblastomas, the most frequent maligt brain tumours
affecting children, comprise at least 4 distinct clinicogenetic subgroups.
Aberrant sonic hedgehog (SHH) signalling is observed in approximately 25% of
tumours and defines one subgroup. Although alterations in SHH pathway genes
(e.g. PTCH1, SUFU) are observed in many of these tumours, high throughput
genomic analyses have identified few other recurring mutations. Here, we have
mutagenised the Ptch+/- murine tumour model using the Sleeping Beauty transposon
system to identify additional genes and pathways involved in SHH subgroup
medulloblastoma development.
RESULTS: Mutagenesis significantly increased medulloblastoma frequency and
identified 17 candidate cancer genes, including orthologs of genes somatically
mutated (PTEN, CREBBP) or associated with poor outcome (PTEN, MYT1L) in the
human disease. Strikingly, these candidate genes were enriched for transcription
factors (p=2x10-5), the majority of which (6/7; Crebbp, Myt1L, Nfia, Nfib, Tead1
and Tgif2) were linked within a single regulatory network enriched for genes
associated with a differentiated neuronal phenotype. Furthermore, activity of
this network varied significantly between the human subgroups, was associated
with metastatic disease, and predicted poor survival specifically within the SHH
subgroup of tumours. Igf2, previously implicated in medulloblastoma, was the
most differentially expressed gene in murine tumours with network perturbation,
and network activity in both mouse and human tumours was characterised by
enrichment for multiple gene-sets indicating increased cell proliferation, IGF
signalling, MYC target upregulation, and decreased neuronal differentiation.
CONCLUSIONS: Collectively, our data support a model of medulloblastoma
development in SB-mutagenised Ptch+/- mice which involves disruption of a novel
transcription factor network leading to Igf2 upregulation, proliferation of
GNPs, and tumour formation. Moreover, our results identify rational therapeutic
targets for SHH subgroup tumours, alongside prognostic biomarkers for the
identification of poor-risk SHH patients. |
What is the prevalence of short QT syndrome? | The prevalence of short QT syndrome is low and varies between 0.01% and 0.1% | BACKGROUND: Long QT syndrome causes ventricular tachyarrhythmias and sudden
death. Recently, a short QT interval has also been shown to be associated with
an increased risk of tachyarrhythmia and sudden death. However, the prevalence
of short QT syndrome is not well-known.
HYPOTHESIS: The aim of this study was to assess the distribution of corrected QT
intervals (QTc) and prevalence of short QT syndrome.
METHODS: This study comprised 12,149 consecutive subjects who received a
consultation at Kanazawa University Hospital, Kanazawa, Japan, and had an
electrocardiogram (ECG) between February 2003 and May 2004. Of these subjects,
1,165 subjects were excluded because of inappropriate ECGs, while the remaining
10,984 subjects had their last-recorded ECGs analyzed.
RESULTS: The QTc values showed a nearly normal distribution (408 +/- 25
msec(1/2)), and were significantly longer in females (412 +/- 24 msec(1/2)) than
in males (404 +/- 25 msec(1/2)) (p < 0.05). Among 5,511 males, 69 subjects
(1.25%) exhibited QTc < 354 msec(1/2) (2 standard deviations [SDs] below the
mean in males), and among 5,473 females, 89 subjects (1.63%) exhibited QTc < 364
msec(1/2) (2 SDs below the mean in females). Only 3 subjects (0.03% in all
subjects and 0.05% in males) exhibited QTc < 300 msec(1/2), however, none had
clinical symptoms of short QT syndrome.
CONCLUSIONS: Short QT syndrome may be very rare. BACKGROUND: Abnormally long and short QT intervals are recognized to be
associated with an increased risk for life-threatening ventricular arrhythmias.
It is therefore important to define the upper and lower border of the normal QT.
OBJECTIVE: The aim of this study was to describe the normal distribution of the
QT interval in a contemporary population of young conscripts and to define long
and short limits of the QT interval.
METHODS: In Switzerland, all young male citizens must undergo compulsory
conscription for the Swiss Army at the age of 18 to 19 years. In every
conscript, an electrocardiogram (ECG) is performed. Retrospectively, 41,767
consecutive ECGs of Swiss citizens who underwent conscription for the army
between March 1, 2004, and July 31, 2006, were analyzed.
RESULTS: The mean QTc Bazett interval was 394 +/- 22 ms. One percent of the
conscripts had a Bazett QTc shorter than 347 ms, and one percent had a Bazett
QTc longer than 445 ms, respectively. None of the subjects presented a QTc
Bazett < 300 ms; the prevalence of a QTc Bazett < 320 ms was 0.02%.
CONCLUSION: The present study shows the distribution of QT intervals in an
unselected young population. Because none of the subjects presented a QTc < 300
ms, it may be concluded that the short QT syndrome is a very rare entity in the
population of young male adults. |
Which sports have a risk for commotio cordis? | Participation in sports such as baseball, football, soccer, cricket, hockey and lacrosse has a risk for commotio cordis. | BACKGROUND: Sudden death from cardiac arrest in a young person may occur during
sports play after a blunt blow to the chest in the absence of structural
cardiovascular disease or traumatic injury (cardiac concussion or commotio
cordis). We studied the clinical features of this apparently uncommon but
important phenomenon.
METHODS: We identified cases from the registries of relevant agencies and
organizations, as well as newsmedia accounts, and developed a clinical profile
of 25 children and young adults, 3 to 19 years of age.
RESULTS: Each victim collapsed with cardiac arrest immediately after an
unexpected blow to the chest, which was usually inflicted by a projectile (such
as a baseball or hockey puck). Incidents took place during organized competitive
sports in 16 cases and in recreational settings at home, at school, or on the
playground in 9. In each instance, the impact to the chest was not judged to be
extraordinary for the sport involved and did not appear to have sufficient force
to cause death. Twelve victims collapsed virtually instantaneously on impact,
whereas 13 remained conscious and physically active for a brief time before
cardiac arrest. Cardiopulmonary resuscitation was administered within about
three minutes to 19 victims, but normal cardiac rhythm could be restored in only
2 (both incurred irreversible brain damage and died shortly thereafter). Seven
victims (28 percent) were wearing some form of protective chest padding.
CONCLUSIONS: We speculate that most sudden deaths related to impact to the chest
(not associated with traumatic injury) are due to ventricular dysrhythmia
induced by an abrupt, blunt precordial blow, presumably delivered at an
electrically vulnerable phase of ventricular excitability. This profile of blunt
chest impact leading to cardiac arrest adds to our understanding of the range of
causes of sudden death on the athletic field and may help in the development of
preventive measures. Commotio cordis due to blunt trauma to the precordium is a rare cause of death
in young athletes, occurring less frequently than all of the other
athletics-related deaths. Several measures, such as the use of safety baseballs
and the use of chest protectors, can help protect young athletes from commotio
cordis. In general, sudden cardiac death in athletes is receiving increasing
attention from the public as a result of recent deaths of high-profile athletes.
Sudden cardiac death, however, is rare, with an estimated 1 out of 200,000 high
school athletes at risk each year. However, the personal, physiological, and
cardiovascular benefits of athletics far outweigh the risks. Therefore, the
message to parents is to allow their children to participate in athletics
because the benefits far outweigh the risks. Over the last few years, the recognised cardiovascular risks of sporting
activities have been extended to include cardiac arrest resulting from
low-energy precordial chest impact produced by projectiles (e.g. baseball) or
bodily contact, in the young, healthy and active athlete [also known as commotio
cordis (CC)]. However, case reports of CC in European medical literature can be
traced back for at least 130 years. CC accounts for a small, but important,
subset of sudden death during sporting activities. It is a devastating
electrophysiological event in the young athlete, and one which has generated
considerable concern, both in the medical profession as well as in the public.
The mechanism of sudden death appears to be caused by ventricular fibrillation,
which occurs when the chest impact is delivered within a narrow, electrically
vulnerable portion of the cardiac cycle, that is, during repolarisation, just
before the peak of the T wave. Resuscitation of these victims is possible with
prompt cardiopulmonary resuscitation and defibrillation. Preventive measures,
such as the use of age-appropriate safety baseballs and suitably designed chest
wall protection, may reduce the risk of sudden death and, thus, make the
athletic field a safer place for young athletes. Although thought to be rare, sudden deaths caused by nonpenetrating chest wall
impact in the absence of structural injury to the ribs, sternum, and heart
(commotio cordis) are reported with increasing frequency. This phenomenon is
described in individuals when they are struck by relatively innocent blows to
the chest wall. Young male athletes aged 5 to 18 years are particularly at risk
for this catastrophe. It has been described after blows to the chest from
baseballs, softballs, hockey pucks, and other objects. Death is usually
instantaneous, and successful resuscitation is uncommon. A recently reported
experimental model provides clues to the mechanisms and inferences for the
prevention and treatment of this devastating condition. This swine model shows
that a) ventricular fibrillation results from low-energy chest wall impacts
during a vulnerable period of repolarization, b) the risk of this event can be
decreased with softer-than-standard baseballs, and c) prompt defibrillation is
crucial for resuscitation to be successful. CONTEXT: Although blunt, nonpenetrating chest blows causing sudden cardiac death
(commotio cordis) are often associated with competitive sports, dangers implicit
in such blows can extend into many other life activities.
OBJECTIVE: To describe the comprehensive spectrum of commotio cordis events.
DESIGN AND SETTING: Analysis of confirmed cases from the general community
assembled in the US Commotio Cordis Registry occurring up to September 1, 2001.
MAIN OUTCOME MEASURE: Commotio cordis event.
RESULTS: Of 128 confirmed cases, 122 (95%) were in males and the mean (SD) age
was 13.6 (8.2) years (median, 14 years; range, 3 months to 45 years); only 28
(22%) cases were aged 18 years or older. Commotio cordis events occurred most
commonly during organized sporting events (79 [62%]), such as baseball, but 49
(38%) occurred as part of daily routine and recreational activities. Fatal blows
were inflicted with a wide range of velocities but often occurred inadvertently
and under circumstances not usually associated with risk for sudden death in
informal settings near the home or playground. Twenty-two (28%) participants
were wearing commercially available chest barriers, including 7 in whom the
projectile made direct contact with protective padding (baseball catchers and
lacrosse/hockey goalies), and 2 in whom the projectile was a baseball
specifically designed to reduce risk. Only 21 (16%) individuals survived their
event, with particularly prompt cardiopulmonary resuscitation/defibrillation
(most commonly reversing ventricular fibrillation) the only identifiable factor
associated with a favorable outcome.
CONCLUSIONS: The expanded spectrum of commotio cordis illustrates the potential
dangers implicit in striking the chest, regardless of the intent or force of the
blow. These findings also suggest that the safety of young athletes will be
enhanced by developing more effective preventive strategies (such as chest wall
barriers) to achieve protection from ventricular fibrillation following
precordial blows. BACKGROUND: There are few epidemiologic studies of catastrophic baseball
injuries.
PURPOSE: To develop a profile of catastrophic injuries in baseball players and
to describe relevant risk factors.
STUDY DESIGN: Retrospective cohort study.
METHODS: The authors reviewed 41 incidents of baseball injuries reported to the
National Center for Catastrophic Sports Injury Research from 1982 until 2002.
RESULTS: There were an estimated 1.95 direct catastrophic injuries per year, or
0.43 injuries per 100,000 participants. The most common mechanisms of injury
were a collision of fielders (9) or of a base runner and a fielder (8), a
pitcher hit by a batted ball (14), and an athlete hit by a thrown ball (4).
Catastrophic injuries included 23 severe head injuries, 8 cervical injuries, 3
cases of commotio cordis, and 2 cases each of a collapsed trachea and facial
fractures. Three athletes sustained a severe head injury and facial fractures.
Ten of the 41 injuries were fatalities.
CONCLUSIONS: Suggestions for reducing catastrophic injuries in baseball include
teaching proper techniques to avoid fielding and baserunning collisions,
protecting the pitcher via a combination of screens and/or helmets with
faceguards, continued surveillance and modifications of the bat and ball,
eliminating headfirst slides, and continued analysis of chest protectors and
automatic external defibrillators for commotio cordis. Blunt precordial blows triggering ventricular fibrillation (commotio cordis)
represent a leading cause of sudden death in young athletes. Attention has
focused on the primary prevention of these tragedies with chest barriers. The
U.S. Commotio Cordis Registry was accessed to determine the likelihood of sudden
death in athletes exposed to precordial blows while wearing chest protectors. Of
182 cases of commotio cordis, 85 (47%) occurred during practice or competition
in organized sports. In 32 of these 85 competitive athletes (38%), fatal chest
blows occurred despite the presence of potentially protective equipment.
Athletes wore standard, commercially available chest barriers made of polymer
foam covered by fabric or hard shells, generally perceived as protective from
arrhythmic consequences of the blows. These events occurred in 4 sports: hockey
(n = 13; 1 goalie), football (n = 10), lacrosse (n = 6; 3 goalies), and baseball
(n = 3; all catchers). Scenarios included the failure of the padding to cover
the precordium so that blows circumvented the protective barrier (n = 25) or
projectiles that struck the chest barrier directly (n = 7). In conclusion, a
significant proportion (about 40%) of sudden deaths reported in young
competitive athletes due to blunt chest blows (commotio cordis) occur despite
the presence of commercially available sports equipment generally perceived as
protective. OBJECTIVE: Athletic field risks associated with blunt, nonpenetrating chest
blows (commotio cordis) are receiving increasing attention, but the epidemiology
of these events is incomplete.
METHODS: We assessed our Sudden Death in Young Athletes Registry, 1980-2008, to
formulate a clinical profile of those sudden deaths attributed to commotio
cordis (and other causes) occurring in competitive lacrosse, the most rapidly
growing youth sport in the United States.
RESULTS: Twenty-three sudden deaths or cardiac arrests were identified in high
school and college lacrosse participants. Ages were 18 +/- 2 years; each athlete
was male. Ten died after blunt precordial blows, including 4 goalies wearing
commercially available chest protectors. Twelve others collapsed because of
presumed or documented cardiovascular disease, including hypertrophic
cardiomyopathy, long QT syndrome, mitral valve prolapse, or ruptured cerebral
aneurysm. The mortality rate associated with lacrosse was 1.46 deaths per
100,000 person-years and was similar to that of other sports including baseball,
basketball, football, and hockey. However, deaths attributed to commotio cordis
were more frequent in lacrosse (0.63 deaths per 100,000 person-years) than in
other sports (P < .02), with the exception of hockey.
CONCLUSIONS: Sudden deaths in competitive lacrosse participants are rare and no
more common than in most other sports. These catastrophic events were caused
disproportionately by commotio cordis and included athletes wearing chest
barriers, thereby underscoring the importance of developing effective chest
protection to create a safer athletic environment for our youth. Commotio cordis is arrhythmia or sudden death from low-impact, blunt trauma to
the chest without apparent heart injury. Ventricular fibrillation is the most
common associated arrhythmia, and heart block, bundle branch block, and
ST-segment elevation are also seen. Commotio cordis occurs most commonly in
baseball but has also been reported in hockey, softball, and several other
sports. Approximately two to four cases are reported each year, but the true
incidence is uncertain. Survival is low, even when resuscitation is performed.
Preventive measures include education of participants and coaches, chest
protection, and softer baseballs. Other considerations include having external
automatic defibrillators and trained personnel at youth sporting events. BACKGROUND: The commotio cordis literature has largely focused on events
occurring in the United States. However, with enhanced public awareness,
commotio cordis has been increasingly recognized internationally as a cause of
cardiac arrest and sudden death due to blunt nonpenetrating chest blows.
OBJECTIVE: This study sought to characterize the demographics of commotio cordis
globally in comparison to the U.S. experience.
METHODS: This study used interrogation of the Commotio Cordis Registry
(Minneapolis, Minnesota).
RESULTS: We report 60 cases of commotio cordis occurring outside the United
States from 19 countries (most commonly the United Kingdom and Canada) on 5
continents and compared these events to 2:3 occuring in the U.S. In the 2
groups, events were largely similar demographically, including frequency of
survival (26% in U.S. vs 25%; P = .84), and the striking male predomice
evident in both groups (i.e., 95%), although non-U.S. victims were somewhat
older (19 ± 13 vs 15 ± 9; P = .002). Not unexpectedly, the groups differed with
baseball/softball and football predomit in the United States (55% of events)
and soccer, cricket, and hockey most common internationally (47% of events).
Notably, the frequency with which soccer participation caused commotio cordis
was much more common than expected, particularly in non-U.S. athletes (20% vs 3%
U.S.; P < .001).
CONCLUSION: Commotio cordis demonstrates a global occurrence, very similar
demographically in the United States and internationally. However, the frequency
with which chest blows from soccer balls caused commotio cordis events
(particularly during sports played internationally) seems to contradict the
prevailing notion that air-filled projectiles convey less risk for ventricular
fibrillation than do those with solid cores (e.g., baseball or lacrosse balls). Commotio Cordis (CC) is the second leading cause of mortality in youth sports.
Impacts occurring directly over the left ventricle (LV) during a vulnerable
period of the cardiac cycle can cause ventricular fibrillation (VF), which
results in CC. In order to better understand the pathophysiology of CC, and
develop a mechanical model for CC, appropriate injury criteria need to be
developed. This effort consisted of impacts to seventeen juvenile porcine
specimens (mass 21-45 kg). Impacts were delivered over the cardiac silhouette
during the venerable period of the cardiac cycle. Four impact speeds were used:
13.4, 17.9, 22.4, and 26.8 m/s. The impactor was a lacrosse ball on an aluminum
shaft instrumented with an accelerometer (mass 188 g-215 g). The impacts were
recorded using high-speed video. LV pressure was measured with a catheter.
Univariate binary logistic regression analyses were performed to evaluate the
predictive ability of ten injury criteria. A total of 187 impacts were used in
the analysis. The criteria were evaluated on their predictive ability based on
Somers' D (D) and Goodman-Kruskal gamma (γ). Injury risk functions were created
for all criteria using a 2-parameter Weibull distribution using survival
analysis. The best criteria for predicting CC were impact force (D=0.52, and
γ=0.52) force*compression (D=0.49, and γ=0.49), and impact power (D=0.49, and
γ=0.49). All of these criteria proved significant in predicting the probability
of CC from projectile impacts in youth sports (p<0.01). Force proved to be the
most predictive of the ten criteria evaluated. CONTENT: Commotio cordis is blunt, nonpenetrating trauma to the chest resulting
in irregular heart rhythm and often leading to sudden death. This article
presents the epidemiology, variables leading to commotio cordis, theories on
predisposing factors, diagnosis, treatment, treatment outcomes, and
return-to-play recommendations.
EVIDENCE ACQUISITION: A PubMed (MEDLINE) search for commotio cordis was
conducted on July 1, 2008, and it yielded 106 results, of which 26 were used for
this review, including experimental models, simulation studies, case analysis
studies, case reports, general recommendation, review articles, and editorials.
RESULTS: There are more than 190 reported cases of commotio cordis in the United
States. Forty-seven percent of reported cases occurred during athletic
participation. Commotio cordis is the second-most common cause of sudden cardiac
death in athletes. Occurrence of commotio cordis is related to time of impact
during the cardiac cycle, direct impact over the heart, the hardness and speed
of the projectile, and the ineffectiveness of chest barriers. As a result, the
US Consumer Product Safety Commission recommends that softer "safety" baseballs
be used for youth baseball. Resuscitation using defibrillation was effective in
only 15% of cases. Resuscitation within 3 minutes resulted in a survival rate of
25% (17 of 68 cases). Survival drops to 3% when resuscitation is delayed beyond
3 minutes. Survival of commotio cordis has risen from 10% to 15% since 2001.
Reduced ventricular ejection fraction has been identified in some commotio
cordis survivors.
CONCLUSION: Preventive measures, such as using soft "safety" balls and making
automated external defibrillators available at sporting venues, can reduce
commotio cordis morbidity and mortality. Chest protector designs can be improved
to enhance protection. Return to play is best left to clinical judgment given
that data are lacking with regard to susceptibility for reoccurrence. |
is there an increase in ultrasound comets after intense exercise? | Strenuous exercise and exercise perfomed in extreme conditions provoke increase in interstitial pulmonary water content as shown by the increased number of ultrasuond comets | Can ultrasound be of any help in the diagnosis of alveolar-interstitial
syndrome? In a prospective study, we examined 250 consecutive patients in a
medical intensive care unit: 121 patients with radiologic alveolar-interstitial
syndrome (disseminated to the whole lung, n = 92; localized, n = 29) and 129
patients without radiologic evidence of alveolar-interstitial syndrome. The
antero-lateral chest wall was examined using ultrasound. The ultrasonic feature
of multiple comet-tail artifacts fanning out from the lung surface was
investigated. This pattern was present all over the lung surface in 86 of 92
patients with diffuse alveolar-interstitial syndrome (sensitivity of 93.4%). It
was absent or confined to the last lateral intercostal space in 120 of 129
patients with normal chest X-ray (specificity of 93.0%). Tomodensitometric
correlations showed that the thickened sub-pleural interlobular septa, as well
as ground-glass areas, two lesions present in acute pulmonary edema, were
associated with the presence of the comet-tail artifact. In conclusion, presence
of the comet-tail artifact allowed diagnosis of alveolar-interstitial syndrome. The "comet-tail" is an ultrasound sign detectable with ultrasound chest
instruments; this sign consists of multiple comet-tails fanning out from the
lung surface. They originate from water-thickened interlobular septa and would
be ideal for nonradiologic bedside assessment of extravascular lung water. To
assess the feasibility and value of ultrasonic comet signs, we studied 121
consecutive hospitalized patients (43 women and 78 men; aged 67 +/- 12 years)
admitted to our combined cardiology-pneumology department (including cardiac
intensive care unit); the study was conducted with commercially available
echocardiographic systems including a portable unit. Transducer frequencies
(range 2.5 to 3.5 MHz) were used. In each patient, the right and left chest was
scanned by examining predefined locations in multiple intercostal spaces.
Examiners blinded to clinical diagnoses noted the presence and numbers of lung
comets at each examining site. A patient lung comet score was obtained by
summing the number of comets in each of the scanning spaces. Within a few
minutes, patients underwent chest x-ray, with specific assessment of
extravascular lung water score by 2 pneumologist-radiologists blinded to
clinical and echo findings. The chest ultrasound scan was obtained in all
patients (feasibility 100%). The imaging time per examination was always <3
minutes. There was a linear correlation between echocardiographic comet score
and radiologic lung water score (r = 0.78, p <0.01). Intrapatient variations (n
= 15) showed an even stronger correlation between changes in echocardiographic
lung comet and radiologic lung water scores (r = 0.89; p <0.01). In 121
consecutive hospitalized patients, we found a linear correlation between
echocardiographic comet scores and radiologic extravascular lung water scores.
Thus, the comet-tail is a simple, non-time-consuming, and reasonably accurate
chest ultrasound sign of extravascular lung water that can be obtained at
bedside (also with portable echocardiographic equipment) and is not restricted
by cardiac acoustic window limitations. Ultrasound lung comet images (ULC) are useful for the noninvasive assessment of
extravascular lung water (EVLW). We investigated the modification of EVLW, its
relation to indices of left ventricular systolic and diastolic function, and
noninvasively determined pulmonary capillary wedge pressure (PCWP) (PCWP = 1.24
ratio of early diastolic mitral inflow velocity to early diastolic velocity of
the mitral annulus [E/Em] + 1.9) at rest and its variation during exercise
echocardiography. A total of 72 patients (mean age 66.4 +/- 8.4 years) with mean
ejection fraction of 41.2 +/- 14.4% underwent symptoms-limited exercise
echocardiography. The sum of the ULC yielded a score of EVLW. The ULC increased
significantly from baseline to postexercise (5.9 +/- 14.9 vs 11 +/- 20.7, P =
.0001). Positive linear correlations were found between baseline ULC score and
baseline ejection fraction (r = -0.37, P = .002), systolic pulmonary artery
pressure (r = 0.69, P = .0001), E/Em (r = 0.70, P = .0001), and estimated PCWP
(r = 0.69, P = .0001). The variation between postexercise and baseline ULC score
correlated significantly with the variation between peak stress and rest PCWP (r
= 0.62, P = .0001), systolic pulmonary artery pressure (r = 0.44, P = .0001),
wall-motion score index (r = 0.30, P = .01), and peak stress E/Em (r = 0.71, P =
.0001), whereas no significant correlations were found between variations of ULC
score and ejection fraction. This study shows that ULC represents a simple way
to assess the presence of excess EVLW. Increased EVLW is associated with
estimated PCWP and indices of left ventricular systolic and diastolic
dysfunction. The additional exercise-induced increase of PCWP, the worsening of
left ventricular diastolic function, and extensive wall-motion abnormalities
correlate with variations of EVLW. BACKGROUND: Ultrasound lung comets (ULCs) detected by chest sonography are a
simple, noninvasive, semiquantitative sign of increased extravascular lung
water. Pulmonary edema may occur in elite apnea divers, possibly triggered by
centralization of blood flow from the periphery to pulmonary vessels. We
assessed the prevalence of ULCs in top-level breath-hold divers after immersion.
METHODS: We evaluated 31 consecutive healthy, top-level, breath-hold divers (10
female, 21 male; age 31 +/- 5 years) participating in a yearly international
apnea diving contest in Sharm-el-Sheik, Egypt, November 1 to 3, 2007. We
performed chest and cardiac sonography with a transthoracic probe (2.5-3.5 MHz,
Esaote Mylab) in all divers, both on the day before and 10 +/- 9 minutes after
immersion. In a subset of 4 divers, chest scan was also repeated at 24 hours
after immersion. ULCs were evaluated on the anterior and posterior chest at 61
predefined scanning sites. An independent sonographer, blind to both patient
identity and status (pre- or post-diving), scored ULCs.
RESULTS: Diving depth ranged from 31 to 112 m. Duration of immersion ranged from
120 to 225 seconds. The ULC score was 0.5 +/- 1.5 at baseline and 13 +/- 21
after diving (P = .012). At individual patient analysis, ULCs appeared in 14
athletes (45%) after diving. Of these 14 athletes, 4 were asymptomatic, 6 showed
aspecific symptoms with transient loss of motor control ("Samba"), 2 had
palpitations with frequent premature ventricular contractions, and 2 had
persistent cough with hemoptysis and pulmonary crackles. In a subset of 4
athletes with post-diving ULCs in whom late follow-up study also was available,
chest sonography findings fully normalized at 24 hours of follow-up.
CONCLUSION: In top-level breath-hold divers, chest sonography frequently reveals
an increased number of ULCs after immersion, indicating a relatively high
prevalence of (often subclinical) reversible extravascular lung water
accumulation. An increasing number of recreational self-contained underwater breathing
apparatus (SCUBA) divers use trimix of oxygen, helium, and nitrogen for dives
deeper than 60 m of sea water. Although it was seldom linked to the development
of pulmonary edema, whether SCUBA diving affects the extravascular lung water
(EVLW) accumulation is largely unexplored.
METHODS: Seven divers performed six dives on consecutive days using compressed
gas mixture of oxygen, helium, and nitrogen (trimix), with diving depths ranging
from 55 to 80 m. The echocardiographic parameters (bubble grade, lung comets,
mean pulmonary arterial pressure (PAP), and left ventricular function) and the
blood levels of the N-terminal part of pro-brain natriuretic peptide (NT-proBNP)
were assessed before and after each dive.
RESULTS: Venous gas bubbling was detected after each dive with mean probability
of decompression sickness ranging from 1.77% to 3.12%. After each dive, several
ultrasonographically detected lung comets rose significantly, which was
paralleled by increased pulmonary artery pressure (PAP) and decreased left
ventricular contractility (reduced ejection fraction at higher end-systolic and
end-diastolic volumes) as well as the elevated NT-proBNP. The number of
ultrasound lung comets and mean PAP did not return to baseline values after each
dive.
CONCLUSIONS: This is the first report that asymptomatic SCUBA dives are
associated with accumulation of EVLW with concomitant increase in PAP,
diminished left ventricular contractility, and increased release of NT-proBNP,
suggesting a significant cardiopulmonary strain. EVLW and PAP did not return to
baseline during repetitive dives, indicating possible cumulative effect with
increasing the risk for pulmonary edema. Recently, an increase in extravascular lung water (EVLW) accumulation with
diminished left ventricular contractility within 60 min after SCUBA diving was
reported. We have observed previously that diving was associated with reduced
diffusing lung capacity for carbon monoxide (DLCO) and arterial oxygen pressure
for up to 60-80 min postdive. Here we investigated whether increased EVLW
persists 2-3h after successive deep dives in a group of seven male divers. The
echocardiographic indices of pulmonary water accumulation (ultrasound lung
comets (ULC)) and left ventricular function, respiratory functional measurements
and arterial oxygen saturation (SaO(2)) were assessed 2-3h post diving, while
venous gas bubbles (VGB) and the blood levels of NT-proBNP and proANP were
analyzed 40 min after surfacing. Spirometry values, flow-volume, DLCO, SaO(2)
and ULC were unchanged after each dive, except for significant increase in ULC
after the second dive. Left ventricular function was reduced, while NT-proBNP
and proANP levels were significantly elevated after majority of dives,
suggesting a cardiac strain. The purpose of the study was to analyze the ultrasound lung comets (ULCs)
variation, which are a sign of extra-vascular lung water. Forty-two healthy
individuals performed breath-hold diving in different conditions: dynamic
surface apnea; deep variable-weight apnea and shallow, face immersed without
effort (static maximal and non-maximal). The number of ULCs was evaluated by
means of an ultrasound scan of the chest, before and after breath-hold diving
sessions. The ULC score increased significantly from baseline after dynamic
surface apnea (p = 0.0068), after deep breath-hold sessions (p = 0.0018), and
after static maximal apnea (p = 0.031). There was no statistically significant
difference between the average increase of ULC scores after dynamic surface
apnea and deep breath-hold diving. We, therefore, postulate that extravascular
lung water accumulation may be due to other factors than (deep) immersion alone,
because it occurs during dynamic surface apnea as well. Three mechanisms may be
responsible for this. First, the immersion-induced hydrostatic pressure gradient
applied on the body causes a shift of peripheral venous blood towards the
thorax. Second, the blood pooling effect found during the diving response
Redistributes blood to the pulmonary vascular bed. Third, it is possible that
the intense involuntary diaphragmatic contractions occurring during the
"struggle phase" of the breath-hold can also produce a blood shift from the
pulmonary capillaries to the pulmonary alveoli. A combination of these factors
may explain the observed increase in ULC scores in deep, shallow maximal and
shallow dynamic apneas, whereas shallow non-maximal apneas seem to be not "ULC
provoking". Pulmonary edema has been reported in breath-hold divers during fish-catching
diving activity. The present study was designed to detect possible increases in
extravascular lung water (EVLW) in underwater fishermen after a competition.
Thirty healthy subjects were studied. They participated in two different 5-h
fish-catching diving competitions: one organized in the winter (10 subjects) and
one organized in the autumn (20 subjects). A questionnaire was used to record
underwater activity and note respiratory problems. An increase in EVLW was
investigated from the detection of ultrasound lung comets (ULC) by chest
ultrasonography. Complementary investigations included echocardiography and
pulmonary function testing. An increase in EVLW was detected in three out of 30
underwater fishermen after the competition. No signs of cardiovascular
dysfunction were found in the entire population and in divers with an increase
in the ULC score. Two divers with raised ULC presented respiratory disorders
such as cough or shortness of breath. Impairment in spirometric parameters was
recorded in these subjects. An increase in EVLW could be observed after a
fish-catching diving competition in three out of 30 underwater fishermen. In two
subjects, it was related to respiratory disorders and impairment in pulmonary
flow. There are several pieces of evidence showing occurrence of pulmonary edema (PE)
in healthy subjects in extreme conditions consisting of extreme psychophysical
demand in normal environment and psychophysical performances in extreme
environment. A combination of different mechanisms, such as mechanical,
hemodynamic, biochemical, and hypoxemic ones, may underlie PE leading to an
increase in lung vascular hydrostatic pressure and lung vascular permeability
and/or a downregulation of the alveolar fluid reabsorption pathways. PE can be
functionally detected by closing volume measurement and lung diffusing capacity
test to different gases or directly visualized by multiple imaging techniques.
Among them chest ultrasonography can detect and quantify the extravascular lung
water, creating "comet-tail" ultrasound artefacts (ULCs) from water-thickened
pulmonary interlobular septa. In this paper the physiopathological mechanisms of
PE, the functional and imaging techniques applied to detect and quantify the
phenomenon, and three models of extreme conditions, that is, ironman athletes,
climbers and breath-hold divers, are described. BACKGROUND: Data regarding the effect of high altitude on heart function are
sparse and conflicting. We aimed to assess the right and left ventricular
responses to altitude-induced hypoxia and the occurrence of subclinical
pulmonary edema.
METHODS: Echocardiography was performed according to protocol on 14 subjects
participating in an expedition in Nepal, at 3 altitude levels: Montreal (30 m),
Namche Bazaar (3450 m), and Chukkung (4730 m). Systematic lung ultrasound was
performed to detect ultrasound lung comets.
RESULTS: Pulmonary artery systolic pressure increased in all subjects between
Montreal and Chukkung (mean 27.4 ± 5.4 mm Hg vs. 39.3 ± 7.7 mm Hg; P < 0.001).
Right ventricular (RV) myocardial performance index (MPI) increased
significantly (0.32 ± 0.08 at 30 m vs. 0.41 ± 0.10 at 4730 m; P = 0.046). A
trend toward deteriorated RV free wall longitudinal strain was observed between
Montreal and Chukkung (-25.9 [5.3%] vs. -21.9 [6.4%]; P = 0.092). The left
ventricular early diastolic inflow velocity/atrial mitral inflow velocity and
early diastolic inflow velocity/mean of the maximal early diastolic mitral
annulus tissue doppler velocities ratios remained unchanged. At 4730 m,
ultrasound lung comets were seen in all subjects except 1. None had clinical
criteria for high-altitude pulmonary edema (HAPE). All altered parameters
normalized after return to sea level.
CONCLUSION: Subclinical HAPE is frequent in healthy lowlander climbers. This is
the first study to document a trend towards decreased RV free wall strain and
MPI increment at high altitude. Whether rising RV MPI is a physiologic adaptive
mechanism to hypoxia or a pathologic response identifying HAPE-susceptible
subjects needs further study. |
Describe Hot water reflex epilepsy. | Hot water epilepsy (HWE) refers to a specific type of reflex epilepsy precipitated by the stimulus of bathing in hot water. Pathogenesis is still unknown and temporal lobe has been thought to take part in the epileptogenesis. HWE can be symptomatic of focal cortical malformation, and few cases were reported. Intermittent clobazam prophylaxis prior to head water bath might be a preferred mode of treatment of pure HWE. | A patient with reflex epilepsy is described, in whom seizures were induced by
bathing in hot water. The literature is reviewed. "Hot water epilepsy" (HWE), precipitated by a bath or shower in hot water, has
been described infrequently in the literature. We report 279 cases of HWE that
were seen between 1980 to 1983 in Bangalore, South India. We found HWE to be
more common in children, with cases more frequent among male than female
patients (2.6:1). Complex partial seizures constituted the main clinical
presentation (67.0%); HWE accounted for 4.4% of all complex partial seizures and
generalize tonic-clonic seizures seen at our center during the 1980-1983 period.
Although prognosis seems favorable 25.4% of our patients developed nonreflex
epilepsy within 1-3 years. They were managed with antiepileptic drugs and the
use of lukewarm water for bathing. BACKGROUND: Hot water epilepsy belongs to the group of reflex epilepsies.
Seizures are provoked by hot water, due to the association of both cutaneous and
heat stimuli. Described mainly in India and Japan, it seems to be rare in Europe
where it occurs in young children.
CASE REPORTS: Five infants aged between 6 months to 2 years had seizures during
bathing with activity arrest, hypotonia and vasoactive modification. Sometimes
clonic movements could be observed. The diagnosis was confirmed by EEG recorded
during bath in the fives cases, with video for two of them. The course of the
seizures and of the psychomotor development were favorable.
CONCLUSION: Hot water epilepsy is a benign epilepsy. Its incidence could be
underestimated because seizures can be confused with febrile convulsions or
vagal fits. Hot water epilepsy is a reflex epilepsy. Seizures are provoked by hot water, and
result from the association of both cutaneous and heat stimuli. Described mainly
in India and Japan, the condition seems to be rare in Europe, where it occurs in
young children. We report five infants aged from 6 months to 2 years. They had
brief seizures during bathing with activity arrest, hypotonia, and vasoactive
modification; clonic movements were observed. A simple treatment-decreasing the
bath temperature-can be sufficient. Sometimes an antiepileptic drug is required.
Seizure course and psychomotor development are favorable. Hot water epilepsy is
a benign form of epilepsy. Its incidence could be underestimated because of
confusion with febrile convulsions, vagal fits, or aquagenic urticaria. We report on monozygotic twins with neonatal onset of daily reflex seizures
triggered by hot water. Video record during the hot water bathing showed
clinical signs consistent with a reflex seizure. The numbers of episodes were
markedly reduced when the mother began bathing the children with reduced
temperature bath water. At the age of 20 months, the twins developed episodes of
paroxysmal disturbances including alternating hemiplegia. These two patients are
the youngest reported cases of reflex hot water seizures, and the only reported
cases in which reflex hot water seizures subsequently manifested episodes of
alternating hemiplegia. We present the case report of a 13-month-old Caucasian toddler with symptoms of
loss of consciousness, central cyanosis and uncontrolled movements of the upper
limbs while taking a warm bath. The diagnosis of hot water epilepsy was
supported by an ictal EEG. Hot water epilepsy, also known as bathing epilepsy or
water-immersion epilepsy is, in the Caucasian population, a rare form of benign
epilepsy, where seizures are provoked by immersion in a hot or even just a warm
bath. This is the first comprehensive video publication of a seizure provoked by
water-immersion in a Caucasian child. [Published with video sequences]. Hot water epilepsy is a reflex or sensory epilepsy in which seizures are
triggered by the stimulus of bathing in hot water. Although there is evidence of
a genetic basis to its etiology, no gene associated with this disorder has so
far been found. In order to identify the genetic locus involved in the
pathophysiology of hot water epilepsy, we performed a genome-wide linkage
analysis in a four-generation family manifesting the disorder in an autosomal
domit manner. Significant linkage was detected on chromosome 4q24-q28, with
the highest two-point LOD score of 3.50 at recombination value (theta) of 0 for
the marker D4S402. Centromere-proximal and centromere-distal boundaries of this
locus were defined by the markers D4S1572 and D4S2277, respectively. The
critical genetic interval spans 22.5 cM and corresponds to about 24 megabases of
DNA. The genes NEUROG2, ANK2, UGT8 and CAMK2D, which are known to be expressed
in human brain, are strong positional candidates and we propose to examine these
and other genes in the locus to identify the causative gene for this intriguing
form of epilepsy. OBJECTIVES: Hot water epilepsy (HWE) or bathing epilepsy is one of the reflex
epilepsies induced by hot water pouring over the head, face, neck, or trunk
during bathing. The aim of this study was to demonstrate the clinical and
electroencephalographic features and the management alternatives of the patients
with HWE.
METHODS: The age of seizure onset, duration of seizure, family history,
interictal and postictal electroencephalography findings, triggering temperature
of water, type of seizure, medication, and follow-up results were evaluated for
each patient.
RESULTS: The mean age at seizure onset was 10.5 years. The mean duration of
seizures was 10 years. Interictal EEG recordings showed focal abnormalities in 4
patients and generalized abnormalities in 3 patients. Only one patient had
normal interictal EEG findings. Among the 8 patients with HWE, 6 had seizures
only during hot bathing, whereas 2 had additional seizures. Seven patients had
generalized tonic-clonic seizures and 1 patient had complex partial seizure
during their hot bathings. The mean triggering temperature of water was
calculated as 41.4 degrees C. The mean duration of follow-up period was 23
months. Five patients became seizure-free during the follow-up period and
seizures persisted in 3 patients. Antiepileptic drugs were given (800 mg/d
carbamazepine for 2 patients and 600 mg/d phenytoin for 1 patient) to these 3
patients and they also became seizure-free during the follow-up period.
CONCLUSIONS: Hot water epilepsy is a benign reflex epilepsy. Lowering water
temperature must be the first step for the treatment. If needed, antiepileptic
drugs should be considered as an additive treatment. Hot water epilepsy is a form of reflex epilepsy in childhood. We report two
children from Saudi Arabia, who presented with seizures following pouring hot
water on their head, while bathing. They were not treated by anti-epileptic
medication. By decreasing the temperature of the water used for bathing, the
seizures were avoided to a large extent in them. This form of epilepsy is
reported to be seldom present in various countries but there are no records of
its presence in Saudi Arabia. Hot water epilepsy (HWE) refers to a specific type of reflex epilepsy
precipitated by the stimulus of bathing in hot water. Pathogenesis is still
unknown and temporal lobe has been thought to take part in the epileptogenesis.
HWE can be symptomatic of focal cortical malformation, and few cases were
reported. This is the third report of HEW in which a parietal malformation has
been observed. Our hypothesis that sensory cortex might be implicated in the
epileptogenic process is corroborated by two previous reports on patients with
HWE and malformation of the parietal cortical development. Measuring neuro-haemodynamic correlates in the brain of epilepsy patients using
EEG-fMRI has opened new avenues in clinical neuroscience, as these are two
complementary methods for understanding brain function. In this study, we
investigated three patients with drug-resistant reflex epilepsy using EEG-fMRI.
Different types of reflex epilepsy such as eating, startle myoclonus, and hot
water epilepsy were included in the study. The analysis of EEG-fMRI data was
based on the visual identification of interictal epileptiform discharges on scalp
EEG. The convolution of onset time and duration of these epilepsy spikes was
estimated, and using these condition-specific effects in a general linear model
approach, we evaluated activation of fMRI. Patients with startle myoclonus
epilepsy experienced epilepsy in response to sudden sound or touch, in
association with increased delta and theta activity with a spike-and-slow-wave
pattern of interictal epileptiform discharges on EEG and fronto-parietal network
activation pattern on SPECT and EEG-fMRI. Eating epilepsy was triggered by sight
or smell of food and fronto-temporal discharges were noted on video-EEG (VEEG).
Similarly, fronto-temporo-parietal involvement was noted on SPECT and EEG-fMRI.
Hot water epilepsy was triggered by contact with hot water either in the bath or
by hand immersion, and VEEG showed fronto-parietal involvement. SPECT and EEG
fMRI revealed a similar fronto-parietal-occipital involvement. From these
results, we conclude that continuous EEG recording can improve the modelling of
BOLD changes related to interictal epileptic activity and this can thus be used
to understand the neuro-haemodynamic substrates involved in reflex epilepsy. A 9-year-old Caucasian boy affected by hot water epilepsy, with positive family
history, experienced complex partial seizures during contact with hot water. A
video-EEG recording was taken while hot water was poured onto his chest. Hot
water epilepsy is rarely described in European countries, where bathing epilepsy
in younger children is more common and often confused with this type of
epilepsy. PURPOSE: To evaluate the role of intermittent prophylaxis with clobazam in the
management of HWE in a long-term prospective study.
MATERIAL AND METHODS: Two hundred and sixty patients [M:F - 194:66] with HWE
were recruited. Patients were divided into: (a) 'HWE alone' (n=198) - received
intermittent clobazam prophylaxis, 1-1½h prior to hot water head bath (group A);
(b) 62 patients (20.4%) with 'HWE with spontaneous seizures were treated with
continuous AEDs along with intermittent clobazam therapy (group B).
RESULTS: Patients (n=198) in group A was followed for mean of 17.6 ± 10.6 months
(range: 3-57). One hundred and forty seven patients (74.2%) had excellent
response with complete seizure freedom with clobazam therapy while 12 (6.1%) had
>75% reduction in seizure frequency. Remaining 39 (19.7%) required additional
standard AED along with clobazam and 18 patients among them developed
spontaneous/unprovoked seizure at follow up of 6.7 ± 4.1 months. Forty five
patients in group B were seizure free while on continuous AEDs.
CONCLUSIONS: Intermittent clobazam prophylaxis prior to head water bath might be
a preferred mode of treatment of pure HWE. Additional AEDs are required if they
have associated non-reflex unprovoked seizure. We studied the anatomical correlates of reflex hot water epilepsy (HWE) using
multimodality investigations viz. magnetic resoce imaging (MRI),
electroencephalography (EEG), and single photon emission computed tomography
(SPECT). Five men (mean age: 27.0 5.8 years) with HWE were subjected to MRI of
brain, video-EEG studies, and SPECT scan. These were correlated with phenotypic
presentations. Seizures could be precipitated in three patients with pouring of
hot water over the head and semiology of seizures was suggestive of temporal
lobe epilepsy. Ictal SPECT showed hyperperfusion in: left medial temporal - one,
left lateral temporal - one, and right parietal - one. Interictal SPECT was
normal in all five patients and did not help in localization. MRI and interictal
EEG was normal in all the patients. The clinical and SPECT studies suggested
temporal lobe as the seizure onset zone in some of the patients with HWE. |
What are the breath test biomarkers of pulmonary tuberculosis | Nitric oxide, urea, volatile organic compounds, hydrogen peroxide and end products of lipid peroxidation are the breath test biomarkers of pulmonary tuberculosis. | Tuberculosis and sarcoidosis represent the granulomatous diseases. The aim of
the study was to compare the markers of oxidative stress: in exhaled breath
condensate (EBC) and in serum of patients with tuberculosis and sarcoidosis.
MATERIAL AND METHODS: 19 patients with active lung tuberculosis and 15 patients
with sarcoidosis were enrolled into the study. As a control served 15 healthy
subjects. Hydrogen peroxide (H2O2) was measured in EBC and the ends products of
lipid peroxidation (TBARs) were assessed in serum.
RESULTS: The concentrations of H202 and TBARs (1022.96+/-186.02 nM and 4.22+/-0.
80 microM, respectively) were significantly higher in patients with tuberculosis
as compared with the controls (398.15+/-37.10 nM and 0.48+/-0.17 microM,
respectively). The patients with sarcoidosis revealed only the significantly
elevated levels of hydrogen peroxide (963.30+/-105.77 nM) in breath condensate.
CONCLUSIONS: It was found that local and systemic oxidative stress were present
in patients with tuberculosis, while in those with sarcoidosis existed only the
local reaction. Recent figures show that tuberculosis (TB) is advancing and killing more than
two million people annually, yet no breakthrough in rapid diagnostics is in
sight. Volatile metabolites of Mycobacterium tuberculosis (MTB) may provide just
that. It is well established that MTB produces nicotinic acid in vitro. We have
converted the free acid into methyl nicotinate and detected statistically
significant differences in the breath of smear positive patients compared with
healthy (smear negative) subjects. BACKGROUND: Volatile organic compounds (VOCs) in breath may contain biomarkers
of active pulmonary tuberculosis derived from the infectious organism
(metabolites of Mycobacterium tuberculosis) and from the infected host (products
of oxidative stress).
METHODS: We analyzed breath VOCs in 226 symptomatic high-risk patients in USA,
Philippines, and UK, using gas chromatography/mass spectroscopy. Diagnosis of
disease was based on sputum culture, smear microscopy, chest radiography and
clinical suspicion of tuberculosis (CSTB). Chromatograms were converted to a
series of 8s overlapping time slices. Biomarkers of active pulmonary
tuberculosis were identified with a Monte Carlo analysis of time-slice alveolar
gradients (abundance in breath minus abundance in room air).
RESULTS: Breath VOCs contained apparent biomarkers of active pulmonary
tuberculosis comprising oxidative stress products (alkanes and alkane
derivatives) and volatile metabolites of M. tuberculosis (cyclohexane and
benzene derivatives). Breath biomarkers identified active pulmonary tuberculosis
with C-statistic (area under curve of receiver operating characteristic)=0.85
(i.e. 85% overall accuracy, sensitivity=84.0%, specificity=64.7%) when sputum
culture, microscopy, and chest radiography were either all positive or all
negative. Employing a single criterion of disease, C-statistic=0.76 (smear
microscopy), 0.68 (sputum culture), 0.66 (chest radiography) and 0.65 (CSTB).
CONCLUSION: A breath test identified apparent biomarkers of active pulmonary
tuberculosis with 85% accuracy in symptomatic high-risk subjects. PURPOSE OF REVIEW: Breath testing has developed over the last 20 years. New
techniques that can identify fingerprints for specific diseases and specific
markers of respiratory pathogens have been applied to breath analysis. This
review discusses the recent advances in breath analysis for the diagnosis of
bacterial and fungal lower respiratory tract infections.
RECENT FINDINGS: The current techniques continue to develop rapidly, but
preconcentration techniques are needed to analyse many target volatile organic
compounds for most systems. Breath testing with an electronic nose is promising
for the diagnosis of tuberculosis (TB), and specific volatiles identifiable by
gas chromatography with mass spectrometry have been identified in breath for
Mycobacterium tuberculosis, Pseudomonas aeruginosa and Aspergillus fumigatus,
but are found at very low concentrations in breath. Contamination from the
environment is an ongoing confounding influence. Exhaled breath condensate (EBC)
is disappointing as a diagnostic sample.
SUMMARY: Careful attention needs to be paid to the sensitivity and specificity
of a technique and confounding from the environment. The role of technologies
such as selected ion flow tube-mass spectrometry is emerging. The electronic
nose requires further validation for TB. The identification of specific
microbial biomarkers aids the quest for improved accuracy. EBC is currently of
limited value. A suite of volatiles have previously been identified as specific markers of
Mycobacterium tuberculosis metabolism in vitro. These markers - methyl
phenylacetate, methyl p-anisate, methyl nicotinate, o-phenylanisole with the
addition of methyl salicylate, may also be derived from other sources and
confound development of a breath test for tuberculosis. To identify potential
sources of these potential biomarkers food products, cosmetics, TB medication,
environmental air and cigarette smoke were analysed for these markers using
solid phase microextraction coupled with Gas Chromatography/Mass Spectrometry.
Breath from healthy subjects, including smokers was also tested. Methyl
salicylate was commonly detected, making this unsuitable as a specific marker
for M. tuberculosis. Methyl nicotinate was detected repeatedly in cigarettes.
Methyl phenylacetate was detected in 1.7% of healthy subjects and
o-phenylanisole in just 1% of healthy breath indicating these may be more
suitable for inclusion in the tuberculosis breath test due to their low
"background" level. These results justify further clinical studies to further
explore these markers as specific indicators of M. tuberculosis infection. BACKGROUND: Nitric oxide (NO), a key macrophage antimycobacterial mediator that
ameliorates immunopathology, is measurable in exhaled breath in individuals with
pulmonary tuberculosis. We investigated relationships between fractional exhale
NO (FENO) and initial pulmonary tuberculosis severity, change during treatment,
and relationship with conversion of sputum culture to negative at 2 months.
METHODS: In Papua, we measured FENO in patients with pulmonary tuberculosis at
baseline and serially over 6 months and once in healthy controls. Treatment
outcomes were conversion of sputum culture results at 2 months and time to
conversion of sputum microscopy results.
RESULTS: Among 200 patients with pulmonary tuberculosis and 88 controls, FENO
was lower for patients with pulmonary tuberculosis at diagnosis (geometric mean
FENO, 12.7 parts per billion [ppb]; 95% confidence interval [CI], 11.6-13.8)
than for controls (geometric mean FENO, 16.6 ppb; 95% CI, 14.2-19.5; P = .002),
fell further after treatment initiation (nadir at 1 week), and then recovered by
6 months (P = .03). Lower FENO was associated with more-severe tuberculosis
disease, with FENO directly proportional to weight (P < .001) and forced
vital-capacity (P = .001) and inversely proportional to radiological score (P =
.03). People whose FENO increased or remained unchanged by 2 months were
2.7-fold more likely to achieve conversion of sputum culture than those whose
FENO decreased (odds ratio, 2.72; 95% CI, 1.05-7.12; P = .04).
CONCLUSIONS: Among patients with pulmonary tuberculosis, impaired pulmonary NO
bioavailability is associated with more-severe disease and delayed mycobacterial
clearance. Measures to increase pulmonary NO warrant investigation as adjunctive
tuberculosis treatments. |
What types of DNA mutations are induced by 2-hydroxy-dATP (2-OH-dATP)? | 2-hydroxy-dATP mainly elicits G:C --> A:T transitions, and, to a lesser extent, G:C --> T:A transversions The induction of G:C --> T:A transversions by 2-OH-dATP indicates the formation of G*2-OH-dATP pairs. 2-OH-dATP also induces tandem (CC --> TT) mutations. Altogether, 2-OH-dATP induces both transition and transvertion mutations, such as A:T --> G:C, A:T --> C:G and G:C --> T:A mutations. | We found that hydroxylation occurs at the C-2 position of adenine by oxygen
radical treatment (Fe2+-EDTA) of dA, dATP, and single- and double-stranded DNA.
This oxidatively damaged base, 2-hydroxyadenine, was produced 3-6-fold and
40-fold less than 8-hydroxyguanine when monomers and polynucleotides,
respectively, were treated. To determine whether the damaged nucleotide,
2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into a growing
DNA, and to reveal the kinds of nucleotides opposite which 2-OH-dATP is
incorporated, calf thymus DNA polymerase alpha and the Klenow fragment of
Escherichia coli DNA polymerase I were used in vitro DAN synthesis in the
presence of 2-OH-dATP. DNA polymerase alpha incorporated the nucleotide opposite
T and C in the DNA template. On the other hand, in an experiment using the
Klenow fragment, incorporation of 2-OH-dATP was observed only opposite T.
Steady-state kinetic studies indicated that incorporation of 2-OH-dATP by DNA
polymerase alpha opposite T was favored over that opposite C by a factor of only
4.5. These results indicate that 2-OH-dATP, an oxidatively damaged nucleotide,
is a substrate for DNA polymerases and is incorporated incorrectly by the
replicative DNA polymerase. The insertion specificities of an oxidized dATP analogue,
2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the
alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the
exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our
previous observation that mammalian DNA polymerase alpha incorporated the
oxidized nucleotide opposite T and C, these two E. coli DNA polymerases
incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state
kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times
more frequently opposite T than opposite G. On the other hand, the incorporation
of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2
orders of magnitude more efficient than that opposite G. These results indicate
that the misinsertion specificity of 2-OH-dATP differs between replicative and
repair-type DNA polymerases, and provide a biochemical basis for the mutations
induced by 2-OH-dATP in E. coli. The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine
5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in
vitro replication system with a HeLa extract. 2-OH-dATP induced mutations in a
dose-dependent manner and elicited substitution and deletion mutations. Of the
substitutions, a G.C-->A.T transition including a tandem (CC-->TT) mutation was
mainly observed. This result agrees with our previous observation that mammalian
DNA polymerase alpha misincorporates the oxidized nucleotide opposite C, but is
in contrast to the finding that 2-OH-dATP elicits G.C-->T.A transversions in
Escherichia coli. This type of mutation was also elicited, but to a lesser
extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the
presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1
protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP
in the replication reaction mixture, and probably in living cells. These results
indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by
the MTH1 protein in mammalian cells. BACKGROUND: We recently found that the Escherichia coli Orf135 protein, a
MutT-type enzyme, hydrolysed 2-hydroxy-dATP (2-OH-dATP), and less efficiently,
8-hydroxy-dGTP.
RESULTS: In this study, we examined the effects of the absence of the orf135
gene. Frequencies of spontaneous and H2O2-induced mutations were two- to
three-fold higher in the orf135- strain than in the wild-type strain. These
mutations include various mutations involving a G:C-->T:A transversion, the same
type of mutation elicited by 2-OH-dATP. Over-expression of the Orf135 protein
suppressed mutations even in the wild-type strain, as well as in the orf135-
strain.
CONCLUSIONS: The mutator phenotype of bacteria lacking the Orf135 protein
suggests that this protein is involved in the suppression of mutations induced
by oxidized deoxynucleotides in vivo and that various MutT-type enzymes
contribute to nucleotide pool sanitization. A 2-substituted purine nucleotide analog, 2-hydroxy-2'-deoxyadenosine
5'-triphosphate (2-OH-dATP), was added to a PCR mixture, to examine its
mutagenic potential. The 2-OH-dATP enhanced the total mutation frequency.
Interestingly, 2-OH-dATP induced both transition and transversion mutations,
including A:T-->G:C, A:T-->C:G and G:C-->T:A mutations. In contrast, other
2-substituted purine nucleotide analogs, 2-aminopurine-2'-deoxyriboside
5'-triphosphate and 2-amino-2'-deoxyadenosine 5'-triphosphate, did not affect
the total mutation frequency. These results suggest that 2-OH-dATP is useful in
random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins,
and for analyses of residues in these biomolecules. The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in
vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G
mutations. However, no direct evidence exists for enhanced mutagenicity of
8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type
and mutT E. coli cells, and mutations of a chromosomal gene were monitored.
8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation
induction in the mutT strain being approximately 6-fold higher than that in the
wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of
the MutT protein, increased the mutation to similar degrees in the two strains.
These results constitute the first evidence that the MutT protein suppresses
mutation by 8-OH-dGTP in vivo. Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA
precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and
carcinogenesis. Here, we report the remarkable nature of human DNA polymerase
eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient
and erroneous manner. The polymerase almost exclusively incorporated
8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of
normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite
template thymine (T), guanine (G), or cytosine (C) at substantial rates. The
synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A
paired with template T, G, or C at the termini were efficiently extended. In
contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A
with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the
template bases. It did not extend the primers having the oxidized bases at the
termini either. We propose that human DNA polymerase eta may participate in
oxidative mutagenesis through the efficient and erroneous incorporation of
oxidized dNTPs during DNA synthesis. The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were
examined using an SV40 origin-dependent in vitro replication system with a HeLa
extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate
(8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were
used in this study. The mutation frequency synergistically increased when the
two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP
enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T -->
C.G transversions. The contribution of the highly error-prone DNA polymerase eta
was unlikely, since similar results were observed with an XP-V cell extract. The
possible involvement of 2-hydroxyadenine in the complementary (template) strand
was excluded on the basis of experiments using plasmids containing
2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP
suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1
protein played the major role in the enhancement. These results highlight the
importance of specific hydrolysis of 8-OH-dGTP for the suppression of its
induced mutation. Aberrant oxidation is a property of many tumor cells. Oxidation of DNA
precursors, i.e., deoxynucleotide triphosphates (dNTPs), as well as DNA is a
major cause of genome instability. Here, we report that human DNA polymerase eta
(h Poleta) incorporates oxidized dNTPs, i.e., 2-hydroxy-2'-deoxyadenosine
5'-triphosphate (2-OH-dATP) and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate
(8-OH-dGTP), into DNA in an erroneous and efficient manner, thereby inducing
various types of mutations during in vitro gap-filling DNA synthesis. When
2-OH-dATP was present at a concentration equal to those of the four normal dNTPs
in the reaction mixture, DNA synthesis by h Poleta enhanced the frequency of
G-to-T transversions eight-fold higher than that of the transversions in control
where only the normal dNTPs were present. When 8-OH-dGTP was present at an
equimolar concentration to the normal dNTPs, it enhanced the frequency of A-to-C
transversions 17-fold higher than the control. It also increased the frequency
of C-to-A transversions about two-fold. These results suggest that h Poleta
incorporates 2-OH-dATP opposite template G and incorporates 8-OH-dGTP opposite
template A and slightly opposite template C during DNA synthesis. Besides base
substitutions, h Poleta enhanced the frequency of single-base frameshifts and
deletions with the size of more than 100 base pairs when 8-OH-dGTP was present
in the reaction mixture. Since h Poleta is present in replication foci even
without exogenous DNA damage, we suggest that h Poleta may be involved in
induction of various types of mutations through the erroneous and efficient
incorporation of oxidized dNTPs into DNA in human cells. |
Which diseases can be treated with Afamelanotide? | Afamelanotide was ivestigated for treatment of erythropoietic protoporphyria, vitiligo, Hailey-Hailey disease, acne vulgaris, polymorphic light eruption, prevention of actinic keratoses in organ transplant recipients and nonmelanoma skin cancer. | Basal cell carcinoma and squamous cell carcinoma are the most frequent types of
cancer in the United States and represent 75 percent and 20 percent,
respectively, of all nonmelanoma skin cancers. Since ultraviolet radiation is
implicated in their development, photoprotection is fundamental in their
prevention. Additional preventive measures include identifying high-risk
individuals for early detection along with using agents, such as retinoids, that
are effective in decreasing the risk of premaligt cells further developing
into carcinomas. Newer agents achieving this goal include perillyl alcohol, T4
endonuclease 5, DL-alpha-tocopherol, and alpha-difluoromethylornithine.
Procedural modalities are currently the standard of treatment, but recent
evidence has consistently shown that newer (nonsurgical) therapies, such as
interferon, imiquimod, retinoids, and 5-fluorouracil, can be used effectively
either as monotherapies or as adjuvants to those surgical modalities for the
treatment of superficial nonmelanoma skin cancers and premaligt lesions.
These newer therapies have achieved significant reductions in morbidity and
mortality. Procedural modalities that have been evolving into important tools
for the treatment of actinic keratosis and nonmelanoma skin cancers include
photodynamic therapy and lasers. Nonsurgical therapies currently proving to be
effective in clinical trials include ingenol mebutate and cyclooxygenase-2
inhibitors. Agents that are showing promising results in early phases of
clinical trials include betulinic acid; hedgehog signaling pathway inhibitors,
such as cyclopamine and GDC-0449; alpha-melanocyte-stimulating hormone analogs,
such as afamelanotide; epidermal growth factor receptor inhibitors, such as
gefitinib and erlotinib; anti-epidermal growth factor receptor monoclonal
antibodies, such as cetuximab and panitumumab; and the 5-fluorouracil prodrug
capecitabine. BACKGROUND: α-Melanocyte-stimulating hormone (α-MSH) is a melanocortin peptide
that increases skin pigmentation during ultraviolet light-mediated tanning. As
α-MSH has been shown to possess anti-inflammatory effects, we assessed the
clinical potential of a superpotent α-MSH analogue, afamelanotide
(Nle(4)-D-Phe(7)-α-MSH), in patients with acne vulgaris, the most common
inflammatory skin disorder.
METHODS: Afamelanotide (16 mg) was given in a phase II open-label pilot study
subcutaneously as a sustained-release resorbable implant formulation to 3
patients with mild-to-moderate facial acne vulgaris. Evaluation included lesion
count, adverse effects and patient-reported outcome. Monitoring of laboratory
parameters included differential blood counts, electrolytes, urine analysis, and
liver and kidney function tests. Skin melanin density was measured by
reflectance spectrophotometry.
RESULTS: The total number as well as the number of inflammatory acne lesions
declined in all patients 56 days after the first injection of afamelanotide.
Life quality as measured by Dermatology Life Quality Index likewise improved in
all 3 patients 56 days after the first injection of afamelanotide. There were no
adverse effects except mild and short-term fatigue in one patient. All patients
experienced increased pigmentation especially on the face. Clinically relevant
changes in laboratory parameters were not detected.
CONCLUSIONS: Afamelanotide appears to have anti-inflammatory effects in patients
with mild-to-moderate acne vulgaris. Future trials are needed to confirm the
anti-inflammatory action of this melanocortin analogue in patients with acne
vulgaris. The tridecapeptide afamelanotide (Scenesse®) is a congener of α-melanocyte
stimulating hormone (α-MSH). Upon binding to the melanocortin 1 receptor (MC1R)
on the surface of pigment cells of the skin, the melanocytes, α-MSH or
afamelanotide trigger the synthesis of cAMP, which stimulates the synthesis of
melanin and therefore induces skin tanning. In a recent trial, afamelanotide
administered as controlled release implants protected erythropoietic
protoporphyria (EPP) patients from sunlight induced phototoxic skin reactions.
Administration of biological therapeutic peptides may elicit unwanted
immunogenic responses in recipients of these products. Although in a previous
study using ELISA technique we excluded any newly developed immunogenicity
during prolonged exposure to afamelanotide, we confirmed the previously
published existence of low titers of antibodies against α-MSH in drug-naïve
individuals that cross-reacted with afamelanotide. In order to investigate
whether such antibodies are neutralizing, i.e. could block the biological effect
of afamelanotide, we developed a cell culture-based bioassay. The basis of our
assay was the measurement of afamelanotide-induced cAMP formation in a strain of
the B16 mouse melanoma cell line, G4F-7, expressing the transfected human MC1R.
Average half-effective concentrations of the natural hormone α-MSH and its
congener afamelanotide were 38.8 ± 10.6 and 10.9 ± 7.17 nM (n=5), respectively.
Neutralizing antibodies would reduce the cAMP formation. Two neutralizing
anti-α-MSH antibodies served as positive controls. cAMP formation in the G4F-7
cells after addition of sera of drug-naïve (n=6) and of drug-exposed EPP
patients (n=17) was significantly lower than after that from healthy volunteers
(n=13). There was no difference between drug-naïve and drug-exposed patients.
Using forskolin as a hormone-independent stimulator of cAMP formation, we
excluded an unspecific interference of EPP sera with cAMP formation. We conclude
that afamelanotide even after prolonged application to EPP patients did not
elicit neutralizing antibodies. Further, the low titer immunoreactivity observed
in sera of some drug-naïve individuals had no effect on the biological activity
of afamelanotide. BACKGROUND: Vitiligo is characterized by depigmented patches of skin due to loss
of cutaneous melanocytes. Many recent studies have demonstrated defects in the
melanocortin system in patients with vitiligo, including decreased circulating
and lesional skin levels of α-melanocyte-stimulating hormone (α-MSH).
Afamelanotide is a potent and longer-lasting synthetic analogue of naturally
occurring α-MSH.
OBSERVATIONS: We describe the preliminary results of 4 patients with generalized
vitiligo who developed repigmentation using afamelanotide in combination with
narrowband UV-B (NB-UV-B) phototherapy. Patients were treated 3 times weekly
with NB-UV-B and starting in the second month received a series of 4 monthly
implants containing 16 mg of afamelanotide. Afamelanotide induced faster and
deeper repigmentation in each case. All patients experienced follicular and
confluent areas of repigmentation within 2 days to 4 weeks after the initial
implant, which progressed significantly throughout treatment. All patients
experienced diffuse hyperpigmentation.
CONCLUSIONS: We propose that afamelanotide represents a novel and potentially
effective treatment for vitiligo. The combined therapy of NB-UV-B and
afamelanotide appears to promote melanoblast differentiation, proliferation, and
eumelanogenesis. Further studies are necessary to confirm these observations. Afamelanotide ([Nle4-D-Phe7]-alpha-MSH) is an analog of alpha-melanocyte
stimulating hormone given as a subcutaneous injection. Afamelanotide is
currently undergoing phase II and III trials in Europe and the US for skin
diseases including vitiligo, erythropoietic protoporphyria, polymorphic light
eruption and prevention of actinic keratoses in organ transplant recipients.
Unregulated analogs and chemicals are being sold online ahead of formal
approval. A number of counterfeit chemicals, 'Melanotans' are being sold for
tanning purposes. Currently, afamelanotide is already on the market in Italy and
Switzerland for patients with erythropoietic protoporphyria. This paper will
review the current literature on this promising compound. BACKGROUND: Hailey-Hailey disease (HHD) is a rare, chronic and recurrent
blistering disorder, which is characterized clinically by erosions occurring
primarily in intertriginous regions, and histologically by suprabasal
acantholysis. Oxidative stress plays a specific role in the pathogenesis of HHD,
by regulating the expression of factors playing an important role in
keratinocyte proliferation and differentiation.
AIM: Given the significance of oxidative stress in HHD, we investigated the
potential effects of the antioxidant properties of an α-MSH analogue,
Nle4-D-Phe7-α-MSH (afamelanotide), in HHD lesion-derived keratinocytes.
RESULTS: Treatment of HHD-derived keratinocytes with afamelanotide contributed
to upregulation of Nrf2 [nuclear factor (erythroid-derived 2)-like 2], a
redox-sensitive transcription factor that plays a pivotal role in redox
homeostasis during oxidative stress. Additionally, afamelanotide treatment
restored the defective proliferative capability of lesion-derived keratinocytes.
Our results show that Nrf2 is an important target of the afamelanotide
signalling that reduces oxidative stress. Because afamelanotide possesses
antioxidant effects, we also assessed the clinical potential of this α-MSH
analogue in the treatment of patients with HHD. In a phase II open-label pilot
study, afamelanotide 16 mg was administered subcutaneously as a
sustained-release resorbable implant formulation to two patients with HHD, who
had a number of long-standing skin lesions. For both patients, their scores on
the Short Form-36 improved 30 days after the first injection of afamelanotide,
and both had 100% clearance of HHD lesions 60 days after the first injection,
independently of the lesion location.
CONCLUSIONS: Afamelanotide is effective for the treatment of skin lesions in
HHD. IMPORTANCE: Narrowband UV-B (NB-UV-B) phototherapy is used extensively to treat
vitiligo. Afamelanotide, an analogue of α-melanocyte-stimulating hormone, is
known to induce tanning of the skin.
OBJECTIVE: To evaluate the efficacy and safety of combination therapy for
generalized vitiligo consisting of afamelanotide implant and NB-UV-B
phototherapy.
DESIGN, SETTING, AND PARTICIPANTS: This study was performed in 2 academic
outpatient dermatology centers and 1 private dermatology practice. We enrolled
men and women 18 years or older with Fitzpatrick skin phototypes (SPTs) III to
VI and a confirmed diagnosis of nonsegmental vitiligo that involved 15% to 50%
of total body surface area. Vitiligo was stable or slowly progressive for 3
months. Patients were randomized to combination therapy (n = 28) vs NB-UV-B
monotherapy (n = 27). After 1 month of NB-UV-B phototherapy, 16 mg of
afamelanotide was administered subcutaneously to the combination therapy group
monthly for 4 months while NB-UV-B phototherapy continued; the other group
continued to receive NB-UV-B monotherapy.
INTERVENTIONS: Narrowband UV-B monotherapy vs combined NB-UV-B phototherapy and
afamelanotide.
MAIN OUTCOMES AND MEASURES: Response on the Vitiligo Area Scoring Index and
Vitiligo European Task Force scoring system.
RESULTS: Response in the combination therapy group was superior to that in the
NB-UV-B monotherapy group (P < .05) at day 56. For the face and upper
extremities, a significantly higher percentage of patients in the combination
therapy group achieved repigmentation, and at earlier times (face, 41.0 vs 61.0
days [P = .001]; upper extremities, 46.0 vs 69.0 days [P = .003]). In the
combination therapy group, repigmentation was 48.64% (95% CI, 39.49%-57.80%) at
day 168 vs 33.26% (95% CI, 24.18%-42.33%) in the NB-UV-B monotherapy group.
Notable adverse events included erythema in both groups and minor infections and
nausea in the combination therapy group. Comparison between Fitzpatrick SPTs
showed patients with SPTs IV to VI in the combination therapy group had
improvement in the Vitiligo Area Scoring Index at days 56 and 84 (P < .05); no
significant difference was noted in patients with SPT III.
CONCLUSIONS AND RELEVANCE: A combination of afamelanotide implant and NB-UV-B
phototherapy resulted in clinically apparent, statistically significant superior
and faster repigmentation compared with NB-UV-B monotherapy. The response was
more noticeable in patients with SPTs IV to VI.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01430195. The application of afamelanotide, an α-melanocyte stimulating hormone agonistic
analogue to protoporphyria, a disease with absolute sunlight-intolerance is
discussed. The clinics, genetics and existing therapies of protoporphyria are
described. The physiological receptor-mediated intracellular signaling of
α-melanocyte stimulating hormone and effects of receptor variants are outlined.
The pharmacological action of afamelanotide and the rationale behind its
application in protoporphyria are given. The results of several Phase II and III
and safety issues are discussed. The trial results were significant, although
the effects were not very large in absolute terms, and the risk-safety profile
is favorable today. Based on the high compliance rate and the excellent
consistency in clinical effectiveness during six years of compassionate use
program in Switzerland, we expect afamelanotide and analogues to become a
prospective therapeutic tool. Moreover, we hope that dosage forms suitable for
children will be developed in future, as children and adolescents suffer most in
protoporphyria. BACKGROUND: Erythropoietic protoporphyria is a severe photodermatosis that is
associated with acute phototoxicity. Patients with this condition have
excruciating pain and a markedly reduced quality of life. We evaluated the
safety and efficacy of an α-melanocyte-stimulating hormone analogue,
afamelanotide, to decrease pain and improve quality of life.
METHODS: We conducted two multicenter, randomized, double-blind,
placebo-controlled trials of subcutaneous implants containing 16 mg of
afamelanotide. Patients in the European Union (74 patients) and the United
States (94 patients) were randomly assigned, in a 1:1 ratio, to receive a
subcutaneous implant containing either afamelanotide or placebo every 60 days (a
total of five implants in the European Union study and three in the U.S study).
The type and duration of sun exposure, number and severity of phototoxic
reactions, and adverse events were recorded over the respective 180-day and
270-day study periods. Quality of life was assessed with the use of validated
questionnaires. A subgroup of U.S. patients underwent photoprovocation testing.
The primary efficacy end point was the number of hours of direct exposure to
sunlight without pain.
RESULTS: In the U.S. study, the duration of pain-free time after 6 months was
longer in the afamelanotide group (median, 69.4 hours, vs. 40.8 hours in the
placebo group; P=0.04). In the European Union study, the duration of pain-free
time after 9 months was also longer in the afamelanotide group than in the
placebo group (median, 6.0 hours vs. 0.8 hours; P=0.005), and the number of
phototoxic reactions was lower in the the afamelanotide group (77 vs. 146,
P=0.04). In both trials, quality of life improved with afamelanotide therapy.
Adverse events were mostly mild; serious adverse events were not thought to be
related to the study drug.
CONCLUSIONS: Afamelanotide had an acceptable side-effect and adverse-event
profile and was associated with an increased duration of sun exposure without
pain and improved quality of life in patients with erythropoietic
protoporphyria. (Funded by Clinuvel Pharmaceuticals and others;
ClinicalTrials.gov numbers, NCT01605136 and NCT00979745.). |
Which is the mechanism used by bacteria to induce tumors in Arabidopsis? | The bacteria Agrobacterium tumefaciens infects Arabidopsis, as well as other plants, and induces the formation of tumors by integrating the transferred-DNA (T-DNA) region of the Ti-plasmid into the plant nuclear genome. | We show that among ecotypes of Arabidopsis, there is considerable variation in
their susceptibility to crown gall disease. Differences in susceptibility are
heritable and, in one ecotype, segregate as a single major contributing locus.
In several ecotypes, recalcitrance to tumorigenesis results from decreased
binding of Agrobacterium to inoculated root explants. The recalcitrance of
another ecotype occurs at a late step in T-DNA transfer. Transient expression of
a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is
deficient in crown gall tumorigenesis, transformation to kanamycin resistance,
and stable GUS expression. This ecotype is also more sensitive to gamma
radiation than is a susceptible ecotype. DNA gel blot analysis showed that after
infection by Agrobacterium, less T-DNA was integrated into the genome of the
recalcitrant ecotype than was integrated into the genome of a highly susceptible
ecotype. Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a
nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The
T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing
plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and
perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid
system was used to screen for proteins interacting with VirD2 and VirE2 to
identify components in Arabidopsis thaliana that interact with the T-complex.
Three VirD2- and two VirE2-interacting proteins were identified. Here we
characterize the interactions of VirD2 with two isoforms of Arabidopsis
cyclophilins identified by using this analysis. The VirD2 domain interacting
with the cyclophilins is distinct from the endonuclease, omega, and the nuclear
localization signal domains. The VirD2-cyclophilin interaction is disrupted in
vitro by cyclosporin A, which also inhibits Agrobacterium-mediated
transformation of Arabidopsis and tobacco. These data strongly suggest that host
cyclophilins play a role in T-DNA transfer. Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans,
induces tumors that produce a family of Amadori-type opines that includes
deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones
mapping to the right of the known oncogenic T-region of pTiChry5 conferred
Amadori opine production on tumors induced by the nopaline strain C58. Sequence
analysis of DNA held in common among these cosmids identified two 25-bp, direct
repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences
are closely related to those of other known T-regions and define a second
T-region of pTiChry5, called T-right (TR), that confers production of the
Amadoriopines. The oncogenic T-left region (TL) was located precisely by
identifying and sequencing the likely border repeats defining this segment. The
two T-regions are separated by approximately 15 kb of plasmid DNA. Based on
these results, we predicted that pKYRT1, a vir helper plasmid derived from
pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with
this hypothesis, transgenic Arabidopsis thaliana plants selected for with a
marker encoded by a binary plasmid following transformation with KYRT1
co-inherited production of the Amadori opines at high frequency. All
opine-positive transgenic plants also contained TR-DNA, while those plants that
lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with
KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic
virus was inserted directly into the vir helper plasmid yielded
kanamycin-resistant transformants at a low but detectable frequency. These
results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA
to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds
of sacB-mediated selection for deletion events. This plasmid, called pKPSF2,
lacks both of the known T-regions and their borders. pKPSF2 failed to transfer
Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an
efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir
helper plasmid pMP90. T-DNA nuclear import is a central event in genetic transformation of plant cells
by Agrobacterium. This event is thought to be mediated by two bacterial
proteins, VirD2 and VirE2, which are associated with the transported T-DNA
molecule. While VirD2 is imported into the nuclei of plant, animal and yeast
cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To
understand better the mechanism of VirE2 action, a cellular interactor of VirE2
was identified and its encoding gene cloned from Arabidopsis. The identified
plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear
import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear
import and Agrobacterium tumorigenicity, participating in early stages of T-DNA
expression. Agrobacterium tumefaciens infects plants and induces the formation of tumors
called "crown galls" by integrating the transferred-DNA (T-DNA) region of the
Ti-plasmid into the plant nuclear genome. Tumors are formed because the T-DNA
encodes enzymes that modify the synthesis of two plant growth hormones, auxin
and cytokinin (CK). Here, we show that a CK biosynthesis enzyme, Tmr, which is
encoded by the Agrobacterium T-DNA region, is targeted to and functions in
plastids of infected plant cells, despite having no typical plastid-targeting
sequence. Evidence is provided that Tmr is an adenosine
phosphate-isopentenyltransferase (IPT) that creates a new CK biosynthesis bypass
by using 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) as a substrate.
Unlike in the conventional CK biosynthesis pathway in plants, trans-zeatin-type
CKs are produced directly without the requirement for P450
monooxygenase-mediated hydroxylation. Consistent with the plastid localization
of Tmr, HMBDP is an intermediate in the methylerythritol phosphate pathway, a
plastid-localized biosynthesis route for universal isoprenoid precursors. These
results demonstrate that A. tumefaciens modifies CK biosynthesis by sending a
key enzyme into plastids of the host plant to promote tumorigenesis. Transformation of plant cells with T-DNA of virulent agrobacteria is one of the
most extreme triggers of developmental changes in higher plants. For rapid
growth and development of resulting tumors, specific changes in the gene
expression profile and metabolic adaptations are required. Increased transport
and metabolic fluxes are critical preconditions for growth and tumor
development. A functional genomics approach, using the Affymetrix whole genome
microarray (approximately 22,800 genes), was applied to measure changes in gene
expression. The solute pattern of Arabidopsis thaliana tumors and uninfected
plant tissues was compared with the respective gene expression profile.
Increased levels of anions, sugars, and amino acids were correlated with changes
in the gene expression of specific enzymes and solute transporters. The
expression profile of genes pivotal for energy metabolism, such as those
involved in photosynthesis, mitochondrial electron transport, and fermentation,
suggested that tumors produce C and N compounds heterotrophically and gain
energy mainly anaerobically. Thus, understanding of gene-to-metabolite networks
in plant tumors promotes the identification of mechanisms that control tumor
development. Agrobacterium tumefaciens causes crown gall disease by transferring and
integrating bacterial DNA (T-DNA) into the plant genome. To examine the
physiological changes and adaptations during Agrobacterium-induced tumor
development, we compared the profiles of salicylic acid (SA), ethylene (ET),
jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the
Arabidopsis thaliana transcriptome. Our data indicate that host responses were
much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101
and that auxin acts as a key modulator of the Arabidopsis-Agrobacterium
interaction. At initiation of infection, elevated levels of IAA and ET were
associated with the induction of host genes involved in IAA, but not ET
signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA
did not. This did not correlate with SA-controlled pathogenesis-related gene
expression in the host, although high SA levels in mutant plants prevented tumor
development, while low levels promoted it. Our data are consistent with a
scenario in which ET and later on SA control virulence of agrobacteria, whereas
ET and auxin stimulate neovascularization during tumor formation. We suggest
that crosstalk among IAA, ET, and SA balances pathogen defense launched by the
host and tumor growth initiated by agrobacteria. Crown gall tumors develop after integration of the T-DNA of virulent
Agrobacterium tumefaciens strains into the plant genome. Expression of the
T-DNA-encoded oncogenes triggers proliferation and differentiation of
transformed plant cells. Crown gall development is known to be accompanied by
global changes in transcription, metabolite levels, and physiological processes.
High levels of abscisic acid (ABA) in crown galls regulate expression of drought
stress responsive genes and mediate drought stress acclimation, which is
essential for wild-type-like tumor growth. An impact of epigenetic processes
such as DNA methylation on crown gall development has been suggested; however,
it has not yet been investigated comprehensively. In this study, the methylation
pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale
as well as at the single gene level. Bisulfite sequencing analysis revealed that
the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls.
Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation,
which inhibited their expression and subsequently crown gall growth. Genome
arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed
a globally hypermethylated crown gall genome, while promoters were rather
hypomethylated. Mutants with reduced non-CG methylation developed larger tumors
than the wild-type controls, indicating that hypermethylation inhibits plant
tumor growth. The differential methylation pattern of crown galls and the stem
tissue from which they originate correlated with transcriptional changes. Genes
known to be transcriptionally inhibited by ABA and methylated in crown galls
became promoter methylated upon treatment of A. thaliana with ABA. This suggests
that the high ABA levels in crown galls may mediate DNA methylation and regulate
expression of genes involved in drought stress protection. In summary, our
studies provide evidence that epigenetic processes regulate gene expression,
physiological processes, and the development of crown gall tumors. The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a
wide range of plant species. The neoplastic growth at the infection sites is
caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A.
tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located
in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production
in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by
the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly
induced by AS under this growth condition but also constitutively expressed and
moderately upregulated by AS at neutral pH 7.0. We found that the promoter
activities and protein levels of several AS-induced vir genes increased in the
tzs deletion mutant, a mutant with decreased tumorigenesis and transient
transformation efficiencies, in Arabidopsis roots. During AS induction and
infection of Arabidopsis roots, the tzs deletion mutant conferred impaired
growth, which could be rescued by genetic complementation and supplementing
exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities
and Vir protein accumulation in both the wild-type and tzs mutant bacteria with
AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in
regulating AS-induced vir gene expression and, therefore, affect bacterial
growth and virulence during A. tumefaciens infection. |
Can cffDNA be used for non-invasive testing? | Yes, cell-free fetal DNA (cffDNA) has made non-invasive prenatal testing possible. | Analysis of cell free fetal DNA (cffDNA) in maternal plasma provides the
opportunity for reliable, timely, safe and cost-effective diagnosis of single
gene disorders. The detection of certain fetal loci using cffDNA and
conventional molecular analytic approaches is possible from 4 weeks gestation.
To date, non-invasive first-trimester analysis for single gene disorders has
been limited by assay sensitivity and specificity, due to the background
maternal DNA. The anticipated ability to enrich the fetal component of cell free
DNA will increase the robustness of tests and permit semi-quantitative analysis,
broadening the scope of testing to include recessive disorders such as cystic
fibrosis. Testing for large-scale mutations might remain limited by the
fragmented nature of cffDNA and, when testing very early in gestation, careful
ultrasound examination will be needed to determine the number of gestational
sacs, because of the risk of discordant twin pregcies. BACKGROUND: Detection of cell-free fetal DNA (cffDNA) in maternal plasma has
given rise to the possibility of new non-invasive approaches for early prenatal
diagnoses. We evaluated the feasibility and accuracy of non-invasive fetal
gender determination using quantitative fluorescent-polymerase chain reaction
(QF-PCR) analysis of circulating cffDNA in the first-trimester maternal plasma.
METHODS: Plasma samples were prospectively collected from 202 singleton
pregcies at 4 to 13 weeks of gestation. Fetal gender was determined by QF-PCR
with the sex-determining region Y (SRY) and amelogenin X/Y (AMELX/Y) genes. The
result was confirmed by fetal karyotyping or phenotype at birth.
RESULTS: Of the 202 pregcies, 162 had pregcy outcomes available and could
be included in our evaluation. The accuracies of AMELX/Y, SRY, and combined
AMELX/Y+SRY analysis for fetal gender determination were 83.3%, 82.1%, and
97.5%, respectively, compared with those of the invasive approach and the fetal
gender outcome at birth (82 males and 80 females). Combined AMELX/Y+SRY analysis
had the highest sensitivity (98.8%) for fetal gender determination with a
specificity of 96.3%. Moreover, fetal gender detection by the combined
AMELX/Y+SRY analysis at 11 to 13 weeks of gestation was 100% correct.
CONCLUSION: Fetal gender determination could be accurately determined from
maternal cffDNA in the first-trimester using QF-PCR analysis of combined
AMELX/Y+SRY. BACKGROUND: The translation of novel genomic technologies from bench to bedside
enjoins the comprehensive consideration of the perspectives of all stakeholders
who stand to influence, or be influenced by, the translational course.
Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA
(cffDNA) circulating in maternal blood is one example of an innovative
technology that promises significant benefits for its intended end users;
however, it is currently uncertain whether it will achieve widespread clinical
implementation. We conducted qualitative interviews with 18 diverse stakeholders
in this domain, including prospective users of the technology and healthcare
personnel, researchers and developers, and experts in social, legal, and
regulatory aspects of genetic technology, and a pilot survey of 62 obstetric
healthcare providers. Analysis of interview and survey data was combined with a
review of the proceedings of a full-day, multidisciplinary conference on the
topic and published scientific and ethics literature surrounding this and other
relevant technologies.
DISCUSSION: We constructed potential pathways for technological implementation,
identified broad stakeholder classes party to these translational processes, and
performed a preliminary assessment of the viewpoints and interrelations among
these diverse stakeholders. Some of the stakeholders whose priorities are
critical to understand and integrate into translation include pregt women and
their families; healthcare providers; scientists, their institutions or
companies, and the funding agencies that support them; regulatory and judicial
bodies; third-party payers; professional societies; educational systems;
disability rights communities; and other representatives from civil society.
Stakeholder interviews, survey findings, and conference proceedings add
complexity to these envisioned pathways and also demonstrate a paramount need to
incorporate an iterative stakeholder analysis early and throughout the
translational endeavor. We believe that the translational framework that we have
developed will help guide crucial future stakeholder mapping and engagement
activities for cffDNA aneuploidy testing and inform novel methods of technology
assessment for other developments in the growing field of genomic medicine.
SUMMARY: Mapping potential pathways for implementation and exploring the
attitudes and interrelations of diverse stakeholders may lead to more effective
translation of a novel method of prenatal aneuploidy testing. The recent release of new, non-invasive prenatal tests for fetal aneuploidy
using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal
testing and has triggered significant commercial interest in the field. Ongoing
research portends the arrival of a wide range of cffDNA tests. However, it is
not yet clear how these tests will be integrated into well-established prenatal
testing strategies in the USA, as the timing of such testing and the degree to
which new non-invasive tests will supplement or replace existing screening and
diagnostic tools remain uncertain. We argue that there is an urgent need for
policy-makers, regulators and professional societies to provide guidance on the
most efficient and ethical manner for such tests to be introduced into clinical
practice in the USA. BACKGROUND: Cell-free fetal DNA (cffDNA) can be detected in maternal blood
during pregcy, opening the possibility of early non-invasive prenatal
diagnosis for a variety of genetic conditions. Since 1997, many studies have
examined the accuracy of prenatal fetal sex determination using cffDNA,
particularly for pregcies at risk of an X-linked condition. Here we report a
review and meta-analysis of the published literature to evaluate the use of
cffDNA for prenatal determination (diagnosis) of fetal sex. We applied a
sensitive search of multiple bibliographic databases including PubMed (MEDLINE),
EMBASE, the Cochrane library and Web of Science.
RESULTS: Ninety studies, incorporating 9,965 pregcies and 10,587 fetal sex
results met our inclusion criteria. Overall mean sensitivity was 96.6% (95%
credible interval 95.2% to 97.7%) and mean specificity was 98.9% (95% CI = 98.1%
to 99.4%). These results vary very little with trimester or week of testing,
indicating that the performance of the test is reliably high.
CONCLUSIONS: Based on this review and meta-analysis we conclude that fetal sex
can be determined with a high level of accuracy by analyzing cffDNA. Using
cffDNA in prenatal diagnosis to replace or complement existing invasive methods
can remove or reduce the risk of miscarriage. Future work should concentrate on
the economic and ethical considerations of implementing an early non-invasive
test for fetal sex. BACKGROUND: Analysis of cell free fetal (cff) DNA in maternal plasma is used
routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination,
fetal rhesus D status and some single gene disorders. True positive results rely
on detection of the fetal target being analysed. No amplification of the target
may be interpreted either as a true negative result or a false negative result
due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A
promoter has been reported as a universal fetal marker to confirm the presence
of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated
maternal sequences are digested leaving hypermethylated fetal sequences
detectable. Complete digestion of maternal sequences is required to eliminate
false positive results.
METHODS: cfDNA was extracted from maternal plasma (n = 90) and digested with
methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A,
SRY and DYS14 was performed by real-time PCR.
RESULTS: Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating
the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were
100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A
and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of
these samples were male at delivery, five had inconclusive results for SRY
analysis and one sample had no amplifiable SRY.
CONCLUSION: Use of this assay for the detection of hypermethylated RASSF1A as a
universal fetal marker has the potential to improve the diagnostic reliability
of NIPD for fetal sex determination and single gene disorders. BACKGROUND: Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in
maternal plasma can predict the fetal RhD type in D negative pregt women. In
Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the
administration of antenatal anti-D prophylaxis only to women who carry an RhD
positive fetus. Prophylaxis reduces the risk of immunization that may lead to
hemolytic disease of the fetus and the newborn. The reliability of predicting
the fetal RhD type depends on pre-analytical factors and assay sensitivity. We
evaluated the testing setup in the Capital Region of Denmark, based on data from
routine antenatal RHD screening.
METHODS: Blood samples were drawn at gestational age 25 weeks. DNA extracted
from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon
7/10. We investigated the effect of blood sample transportation time (n = 110)
and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total
DNA. We compared two different quantification methods, the delta Ct method and a
universal standard curve. PCR pipetting was compared on two systems (n = 104).
RESULTS: The cffDNA level was unaffected by blood sample transportation for up
to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C
during transport. The universal standard curve was applicable for cffDNA
quantification. Identical levels of cffDNA were observed using the two automated
PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median
gestational age of 25 weeks (range 10-39, n = 1317).
CONCLUSION: The setup for real-time PCR-based, non-invasive prenatal testing of
cffDNA in the Capital Region of Denmark is very robust. Our findings regarding
the transportation of blood samples demonstrate the high stability of cffDNA.
The applicability of a universal standard curve facilitates easy cffDNA
quantification. NIFTY (Non-invasive Fetal Trisomy Test) is a non-invasive prenatal test which is
used for diagnosing fetal trisomy. The test is based on the analysis of cell
free fetal DNA (cffDNA) present in the plasma and serum of a pregt woman.
NIFTY allows to detect fetal trisomy of chromosomes 13, 18, 21, X and Y and also
X monosomy. Abnormal NIFTY results still need to be verified using other
diagnostic techniques. However the sensitivity of NIFTY for trisomy 21, 18 and
13 is estimated at 99%, 97% and 79% respectively with false positive rate for
all examined trisomies and X monosomy of < 1%. NIFTY is currently available in
Poland as a commercial service, used as a good screening test for common
trisomies (apart from ultrasound and biochemical tests) in the case of patient
anxiety and in situation when the patient does not consent to invasive prenatal
diagnostic tests. The sensitivity and specificity of NIFTY will most likely be
improved as laboratory methods develop, and after a sufficiently large group of
pregt patients has been tested. Therefore, this test may soon become the
primary diagnostic tool for common trisomies, allowing to avoid invasive
prenatal testing in this indication. With high probability cffDNA obtained from
the serum of pregt women will also be used with time in the diagnosis of
fetal structural chromosomal aberrations and other genetic changes. The aim of
our study is to present a new diagnostic method. |
List features of the Perry syndrome. | Perry syndrome is a familial parkinsonism associated with central hypoventilation, mental depression, and weight loss. | Perry syndrome consists of early-onset parkinsonism, depression, severe weight
loss and hypoventilation, with brain pathology characterized by TDP-43
immunostaining. We carried out genome-wide linkage analysis and identified five
disease-segregating mutations affecting the CAP-Gly domain of dynactin (encoded
by DCTN1) in eight families with Perry syndrome; these mutations diminish
microtubule binding and lead to intracytoplasmic inclusions. Our findings show
that DCTN1 mutations, previously associated with motor neuron disease, can
underlie the selective vulnerability of other neuronal populations in distinct
neurodegenerative disorders. A patient with a mood disorder and a Parkinsonian syndrome with frontal
cognitive impairment thought to resemble progressive supranuclear palsy defied
precise diagnosis until the development of respiratory compromise, prompting
consideration of the diagnosis of Perry syndrome. A mutation in the dynactin 1
gene confirmed the diagnosis. Few examples of this disorder, characterised by
depression, Parkinsonism, and respiratory insufficiency, have been reported but
it may be more commonly recognised with the availability of genetic testing.
Perry syndrome needs to be considered in the differential diagnosis of
Parkinsonism, particularly in autosomal domit pedigrees. Diagnosis early in
the disease course may facilitate monitoring and prompt intervention to avoid
potentially fatal respiratory failure. BACKGROUND: Depression, parkinsonism, and hypoventilation (Perry syndrome) or
familial motor neuron disease have been linked to mutations in dynactin
P150(Glued) (DCTN1).
METHODS: We employed genealogic, clinical, neurologic, and MRI investigations,
as well as analysis of genes implicated in parkinsonism. Cellular transfection,
immunocytochemistry, and immunoprecipitation analysis of wild-type (WT) and
mutant DCTN1 were also performed.
RESULTS: A novel heterozygous mutation, DCTN1 c.156T>G, encoding p.Phe52Leu,
segregates with parkinsonism in a Japanese family. The substitution was not
observed in affected probands with familial parkinsonism or control subjects and
is evolutionarily conserved. In contrast to Perry syndrome, affected carriers
have late-onset disease and slower progression, with frontotemporal atrophy
revealed by MRI. In vitro studies suggest the mutant protein has impaired
microtubule binding, compared to WT dynactin p150(Glued) .
CONCLUSIONS: DCTN1 mutations may contribute to disparate neurodegenerative
diagnoses, including familial motor neuron disease, parkinsonism, and
frontotemporal atrophy, and further studies of dynactin-mediated cargo transport
may prove insightful. |
Which is the main calcium binding protein of the sarcoplasmic reticulum? | Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca(2+ )in the lumen of the sarcoplasmic reticulum (SR). | Calsequestrin is a calcium-binding protein known to sequester calcium
accumulated in the sarcoplasmic reticulum (SR) of muscle cells during
relaxation. In the present study, we used affinity-purified antibodies to
chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog
myocardium. Like previously identified cardiac calsequestrins, it is enriched in
cardiac microsomes, it is enriched by biochemical procedures previously used to
purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift
in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal
amino acid sequence of this 60,000-Da immunoreactive protein purified by fast
protein liquid chromatography was identical to that of rabbit skeletal and
canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to
the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was
localized in discrete foci present at the periphery but absent from the central
regions of frog ventricular myocytes as determined by immunofluorescence
labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin
was confined to the lumen of two structurally distinct regions of the SR, where
it was localized in the subsarcolemmal region of the myofibers. One of these
appeared to correspond to the terminal SR previously reported to be closely
apposed to the sarcolemma of frog myofibers. The other region, although close to
the sarcolemma, was not physically joined to it and appeared to correspond to
corbular SR. It generally is believed that frog cardiac SR does not provide
activator Ca2+ required for excitation-contraction coupling. However, the
identification of a calsequestrin isoform very similar to mammalian cardiac
calsequestrin that is confined to specialized regions of frog cardiac SR lends
support to the idea that frog cardiac SR has the ability to store Ca2+ and thus
function in some capacity in frog cardiac muscle contraction. Calsequestrin is the major calcium-binding protein of cardiac and skeletal
muscles whose function is to sequester Ca(2+ )in the lumen of the sarcoplasmic
reticulum (SR). Here we describe the identification and functional
characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows
moderate similarity (50% similarity, 30% identity) to rabbit skeletal
calsequestrin. Unlike mammals, which have two different genes encoding cardiac
and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene
in the C. elegans genome. We show that csq-1 is highly expressed in the
body-wall muscles, beginning in mid-embryogenesis and maintained through the
adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic
membranes surrounding sarcomeric structures, in the regions where ryanodine
receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization,
suggesting that the two possibly interact in vivo. Genetic analyses of
chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential
for initiation of embryonic muscle formation and contraction. Furthermore,
double-stranded RNA injection resulted in animals completely lacking CSQ-1 in
body-wall muscles with no observable defects in locomotion. These findings
suggest that although CSQ-1 is one of the major calcium-binding proteins in the
body-wall muscles of C. elegans, it is not essential for body-wall muscle
formation and contraction. Mutations in human cardiac calsequestrin (CASQ2), a high-capacity
calcium-binding protein located in the sarcoplasmic reticulum (SR), have
recently been linked to effort-induced ventricular arrhythmia and sudden death
(catecholaminergic polymorphic ventricular tachycardia). However, the precise
mechanisms through which these mutations affect SR function and lead to
arrhythmia are presently unknown. In this study, we explored the effect of
adenoviral-directed expression of a canine CASQ2 protein carrying the
catecholaminergic polymorphic ventricular tachycardia-linked mutation D307H
(CASQ2(D307H)) on Ca2+ signaling in adult rat myocytes. Total CASQ2 protein
levels were consistently elevated approximately 4-fold in cells infected with
adenoviruses expressing either wild-type CASQ2 (CASQ2(WT)) or CASQ2(D307H).
Expression of CASQ2(D307H) reduced the Ca2+ storing capacity of the SR. In
addition, the amplitude, duration, and rise time of macroscopic I(Ca)-induced
Ca2+ transients and of spontaneous Ca2+ sparks were reduced significantly in
myocytes expressing CASQ2(D307H). Myocytes expressing CASQ2(D307H) also
displayed drastic disturbances of rhythmic oscillations in [Ca2+]i and membrane
potential, with signs of delayed afterdepolarizations when undergoing periodic
pacing and exposed to isoproterenol. Importantly, normal rhythmic activity was
restored by loading the SR with the low-affinity Ca2+ buffer, citrate. Our data
suggest that the arrhythmogenic CASQ2(D307H) mutation impairs SR Ca2+ storing
and release functions and destabilizes the Ca2+-induced Ca2+ release mechanism
by reducing the effective Ca2+ buffering inside the SR and/or by altering the
responsiveness of the Ca2+ release channel complex to luminal Ca2+. These
results establish at the cellular level the pathological link between CASQ2
mutations and the predisposition to adrenergically mediated arrhythmias observed
in patients carrying CASQ2 defects. Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein
segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum
(SR). The physiological role of CS in controlling calcium release from the SR
depends on both its intrinsic properties and its localization. The mechanisms of
CS targeting were investigated in skeletal muscle fibers and C2C12 myotubes, a
model of SR differentiation, with four deletion mutants of epitope
(hemagglutinin, HA)-tagged CS: CS-HA24NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a
double mutant of the NH2 terminus and domain III. As judged by
immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA
mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did
not segregate to TC. As shown by subfractionation of microsomes derived from
transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR
whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by
immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not
sorted to developing SR. Condensation competence, a property referable to CS
oligomerization, was monitored for the several CS-HA mutants in C2C12 myoblasts,
and only CS-HA3D was found able to condense. Together, the results indicate that
1) there are at least two targeting sequences at the NH2 terminus and domain III
of CS, 2) SR-specific target and structural information is contained in these
sequences, 3) heterologous interactions with junctional SR proteins are relevant
for segregation, 4) homologous CS-CS interactions are involved in the overall
targeting process, and 5) different targeting mechanisms prevail depending on
the stage of SR differentiation. 1. Many biological processes that are governed by intracellular calcium signals
rely on intracellular stores, which provide a reliable, controlled release of
calcium into the cytoplasm. Calcium release through the ryanodine receptor
(RyR), the main ion channel in the sarcoplasmic reticulum (the calcium store in
muscle) is the key determit of muscle force. 2. Calsequestrin, the main
calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for
release through the RyR and acts as a luminal calcium sensor for the channel via
its interactions with triadin and junctin. Until recently, how calsequestrin
communicated the store Ca(2+) load to the RyR remained unknown. 3. Calsequestrin
1 (skeletal calsequestrin) has been shown to both inhibit and activate the
skeletal RyR1, dependent on whether it's bound to the RyR1 directly or
indirectly via anchoring proteins. 4. The phosphorylation status of
calsequestrin 1 is deemed important: it influences the Ca(2+) binding capacity
of calsequestrin, the way in which calsequestrin 1 regulates the RyR1 and how
calsequestrin 1 interacts with the key anchoring protein junctin. 5. In skeletal
muscle, junctin plays a more critical role than triadin in the mechanism that
controls Ca(2+) release from the sarcoplasmic reticulum. 6. The close
relationship between altered expression and dysfunction of calsequestrin in
several skeletal and cardiac disorders highlights the critical role that
calsequestrin plays in maintaining Ca(2+) homeostasis and regulation of muscle
contraction. Calsequestrin (CASQ) is the major component of the sarcoplasmic reticulum (SR)
lumen in skeletal and cardiac muscles. This calcium-binding protein localizes to
the junctional SR (jSR) cisternae, where it is responsible for the storage of
large amounts of Ca(2+), whereas it is usually absent, at least in its
polymerized form, in the free SR. The retention of CASQ inside the jSR is due
partly to its association with other jSR proteins, such as junctin and triadin,
and partly to its ability to polymerize, in a high Ca(2+) environment, into an
intricate gel that holds the protein in place. In this work, we shed some light
on the still poorly described in situ structure of polymerized CASQ using
detailed EM images from thin sections, with and without tilting, and from
deep-etched rotary-shadowed replicas. The latter directly illustrate the
fundamental network nature of polymerized CASQ, revealing repeated nodal points
connecting short segments of the linear polymer. |
Is the UGT1A1*28 polymorphism associated with irinotecan response in Caucasians? | Yes, it has been shown that the polymorphism UGT1A1*28 is associated with irinotecan response in Caucasians. | The uridine diphosphate glucuronosyltransferase (UGT) 1A1 and 1A9 isoforms are
involved in the phase II biotransformation of the irinotecan metabolite, SN-38.
Recently, several variants in the UGT1A1 and UGT1A9 genes have been described
with altered functionality in vitro. The aim of this study was to evaluate the
functional consequence of the UGT1A1(TA)(7)TAA (UGT1A1(*)28), UGT1A9 766G>A
(D256N; UGT1A9(*)5), and UGT1A9 98T>C (M33T; UGT1A9(*)3) variants in Caucasian
patients treated with irinotecan. Pharmacokinetic studies were performed after
the first course of irinotecan in 47 males and 47 females. The mean (SD) area
under the curves (AUCs) of irinotecan and SN-38 were 20,348 +/- 6466 ng x h/mL
and 629 +/- 370 ng x h/mL, respectively, which is in line with earlier findings.
For UGT1A9(*)5,novariant alleles were observed, whereas for UGT1A9(*)3, 1
patient with the variant allele was found (allele frequency, 0.633%). The
distribution of the UGT1A1(*)28 variant showed 44 wild-type patients (Wt), 37
heterozygotes (Het), and 5 homozygotes (Var). The median AUC ratio of SN-38G to
SN-38 was significantly reduced in carriers of the variant UGT1A1(*)28 allele
(7.00 [Wt] vs. 6.26 [Het] vs. 2.51 [Var]; p =.022). It is concluded that UGT1A9
functional variants are rare in Caucasians and likely to be clinically
insignificant in irinotecan regimens. Screening for the UGT1A1(*)28 polymorphism
may identify patients with altered SN-38 pharmacokinetics. The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase is
responsible for glucuronidation and detoxification of SN-38, the active
metabolite of irinotecan. The presence of an additional TA repeat in the TATA
sequence of the UGT1A1 promoter leads to a significant decrease in SN-38
glucuronidation. Patients with the UGT1A1 (TA)7 allele are more likely to
experience severe neutropenia and diarrhea following irinotecan chemotherapy. We
assessed the distribution of the UGT1A1 (TA)n polymorphism in healthy male and
female US residents of European and Asian descent. We used a fluorescent
polymerase chain reaction-based assay to detect UGT1A1 (TA)n polymorphisms in
138 healthy volunteers (56 Caucasians, 37 Chinese, 37 Filipino and eight
Japanese) between the ages of 18 and 65 years. The chi-test was used to assess
between-group differences in the distribution of UGT1A1 (TA)n genotypes. The
UGT1A1 (TA)6/6 genotype was significantly more common in Asians than in
Caucasians (76 vs. 46%), whereas the (TA)6/7 (39 vs. 20%) and (TA)7/7 (13 vs.
5%) genotypes were more common in Caucasians than in Asians. Genotype
distributions did not differ significantly between men and women in either
group. The UGT1A1 (TA)5/5 genotype was detected in one Caucasian woman. In
conclusion, consistent with previous reports, the UGT1A1 (TA)7/7 genotype was
significantly more common in Caucasians than in Asians. UGT1A1 (TA)n/n genotype
distribution did not vary with sex in individuals of European or Asian descent. Irinotecan is widely used in the treatment of colorectal, gastric, and lung
cancers. However, adverse drug reactions such as severe diarrhea and neutropenia
limit the dose of this drug. Irinotecan is metabolized by carboxylesterase to
form an active metabolite, 7-ethyl-10-hydroxycamptothecin(SN-38), which in turn
is subsequently conjugated by UGT-glucuronosyltransferase 1A1(UGT1A1)to yield an
inactive form, SN-38 glucuronide(SN-38 G). The UGT1A1 gene polymorphisms
contribute to the individual variation in adverse events among patients
administered irinotecan. However, the distribution of polymorphisms shows large
interethnic differences. The distribution of UGT1A1*28 greatly differs between
Caucasians and Japanese; the frequency of UGT1A1*28 is high in Caucasians,
whereas it is low in Asians including Japanese. Recently, it has been
demonstrated that genetic variants of UGT1A1*6 in addition to UGT1A1*28 are
associated with the occurrence of adverse events in irinotecan chemotherapy in
Asians. This review summarizes recent studies to outline the role of UGT1A1*28
and UGT1A1*6 for irinotecan-induced adverse drug reaction in Japanese cancer
patients. Gilbert's syndrome causes mild, unconjugated hyperbilirubinemia and is present
in approximately 10% of the Caucasian population. The basis of the disorder is a
70% reduction in bilirubin glucuronidation catalyzed by the
UDP-glucuronosyltransferase 1A1 (UGT1A1), which, in Caucasians, is the result of
a homozygous TA insertion into the promoter region of the UGT1A1 gene
(UGT1A1*28). Homozygous carriers of UGT1A1*28 as well as those with additional
UGT1A variants can suffer from severe irinotecan toxicity or jaundice during
treatment with the protease inhibitor atazanavir. UGT1A1*28 genotyping
identifies patients at risk for drug toxicity and can increase drug safety by
dose individualization. Rapid and facile UGT1A1*28 genotyping is therefore of
great clinical importance. Two hundred ninety-one patients with suspected
Gilbert's syndrome were genotyped using the TaqMan 5'nuclease assay with minor
groove binder-non fluorescent quench probes; results were confirmed by direct
sequencing. Ninety-six patients (33%) were homozygous for UGT1A1*28, which was
verified by direct sequencing of a different PCR product showing 100%
concordance with the TaqMan PCR results. We describe a novel UGT1A1*28
genotyping method that employs allelic discrimination by TaqMan PCR. This assay
provides a rapid, high-throughput, and cost-effective method for Gilbert's
syndrome genotyping, which is of value for pretreatment screening of potential
irinotecan toxicity. The method utilizes a technological platform that is widely
used in clinical practice and could therefore be easily adapted for routine
clinical applications. BACKGROUND: For genetic polymorphisms known to alter drug effect or safety,
regulatory authorities can tap into population genomic databases and other
sources of allele and genotype distribution data to make a more informed
decision about the anticipated impact of such variants on the main ethnic groups
in a country's population.
OBJECTIVE: The aim of this short communication is to describe how the Singapore
Health Sciences Authority (HSA) made use of allele and genotype distributions in
the main ethnic groups in Singapore (Chinese, Malay, Indian) and population
genetic tools to compare with North American Caucasians and Japanese.
METHODS: Published papers and publicly accessible genomic databases were
searched up to August 2009 to obtain allele and genotype frequencies for
UGT1A1*6 and *28, two common variants of UGT1A1, a gene that encodes for a key
enzyme in the pathway of irinotecan metabolism. These variants are associated
with greater risk of serious toxicity.
RESULTS: In Singapore, the combined prevalence of three high-risk genotypes,
UGT1A1*6/*6, *6/*28 and *28/*28, is 9.7% in Chinese, 5.0% in Malays and 18.7% in
Indians, compared with 11.5% in North American Caucasians and 8.1% in Japanese.
Indians are at an elevated risk of irinotecan-induced neutropenia associated
with UGT1A1*28 compared with Chinese and Japanese, and at an even higher risk
compared with North American Caucasians. On the other hand, Chinese and Japanese
are at an elevated risk of irinotecan-induced neutropenia associated with
UGT1A1*6 relative to Indians in Singapore or North American Caucasians.
Population genotype data were the basis for the HSA to request revision of the
package insert from manufacturers of irinotecan products. Moreover, the data
provided the impetus for the HSA to publicize the availability of UGT1A1 genetic
testing at the National Cancer Centre.
CONCLUSION: With the growing volume of genomic data and pharmacogenomic
associations, a regulatory authority is now able to more readily utilize
population genetic information and tools to supplement evaluations of drug
products pertinent to the country's ethnic demography. Topoisomerase I (TOP-I) mutations have been shown to be correlated to irinotecan
resistance in vitro. However, the prevalence of TOP-I germline mutations has yet
to be systematically elucidated. On the other hand, polymorphisms of UGT1A1 have
been shown to be associated with CPT-11 toxicity in clinical situations. The
primary aim of this study was to investigate the prevalence of mutations in the
TOP-I exons associated with CPT-11 resistance, including untreated cancer
tissue. A secondary aim was to confirm the less frequent UGT1A1*28 and more
frequent UGT1A1*6 in individuals of Asian descent compared to Caucasians and
individuals of African descent. The prevalence of 5 reported TOP-I mutations in
exons was investigated in volunteers (n=236) using DNA sequencing of the PCR
products. The prevalence of TOP-I mutations in untreated lung cancer tissues
(n=16) was also investigated. Additionally, 3 UGT1A1 polymorphisms, UGT1A1*6,
*27 and *28, were investigated in volunteers (n=126). There were no mutations of
TOP-I in any of the 236 subjects or in the untreated lung tissues. Among
128 subjects, the distribution of homozygous polymorphisms of UGT1A1 was:
UGT1A1*28 in 3 (2.4%) and UGT1A1*6 in 4 (3.2%) subjects, and co-occurrence of
heterozygous polymorphisms for both UGT1A1*6 and UGT1A1*28 in 4 (3.2%) subjects,
and for UGT1A1*27 and UGT1A1*28 in 1 subject (0.8%). The Hardy-Weinberg
deviation test showed there was no significant deviation from the equilibrium,
and the association analysis indicated no significant linkage between UGT1A1*6
and UGT1A1*28. In conclusion, TOP-I genetic mutations correlated to CPT-11
resistance were not detected in any of the subjects and untreated lung cancer
tissues. Less frequent UGT1A1*28 and more frequent UGT1A1*6 were confirmed in
East Asian individuals compared to Caucasians and individuals of African
descent. Linkage disequilibrium was not detected between UGT1A1*6 and UGT1A1*28. BACKGROUND: Whether UGT1A1*28 genotype is associated with clinical outcomes of
irinotecan (IRI)-based chemotherapy in Colorectal cancer (CRC) is an important
gap in existing knowledge to inform clinical utility. Published data on the
association between UGT1A1*28 gene polymorphisms and clinical outcomes of
IRI-based chemotherapy in CRC were inconsistent.
METHODOLOGY/PRINCIPAL FINDINGS: Literature retrieval, trials selection and
assessment, data collection, and statistical analysis were performed according
to the PRISMA guidelines. Primary outcomes included therapeutic response (TR),
progression-free survival (PFS) and overall survival (OS). We calculated odds
ratios (OR) and hazard ratios (HR) with 95% confidence intervals (CI). Twelve
clinical trials were included. No statistical heterogeneity was detected in
analyses of all studies and for each subgroup. Differences in TR, PFS and OS for
any genotype comparison, UGT1A1*28/*28 versus (vs) UGT1A1*1/*1 (homozygous
model), UGT1A1*1/*28 vs UGT1A1*1/*1 (heterozygous model), and UGT1A1*28/*28 vs
all others (recessive model, only for TR) were not statistically significant.
IRI dose also did not impact upon TR and PFS differences between UGT1A1 genotype
groups. A statistically significant increase in the hazard of death was found in
Low IRI subgroup of the homozygous model (HR = 1.48, 95% CI = 1.06-2.07; P =
0.02). The UGT1A1*28 allele was associated with a trend of increase in the
hazard of death in two models (homozygous model: HR = 1.22, 95% CI = 0.99-1.51;
heterozygous model: HR = 1.13, 95% CI = 0.96-1.32). These latter findings were
driven primarily by one single large study (Shulman et al. 2011).
CONCLUSIONS/SIGNIFICANCE: UGT1A1*28 polymorphism cannot be considered as a
reliable predictor of TR and PFS in CRC patients treated with IRI-based
chemotherapy. The OS relationship with UGT1A1*28 in the patients with lower-dose
IRI chemotherapy requires further validation. A meta-analysis in Caucasians was conducted to investigate the possible
association of uridine diphosphate glucuronosyltransferase (UGT) 1A1 gene
polymorphisms with irinotecan (IRI)-induced neutropenia and diarrhoea in
colorectal cancer (CRC). We searched PubMed and Embase until May 2012 to
identify eligible studies, extracted data, assessed methodological quality, and
performed statistical analysis using REVMAN 5.1 and R software. Subgroups
meta-analyses were performed in groups representing different IRI combination
regimens and IRI doses. Sixteen trials were included. UGT1A1*28/*28 genotype was
associated with more than fourfold (odds ratio (OR)=4.79, 95% confidence
intervals (CI): 3.28-7.01; P<0.00001) and threefold (OR=3.44, 95% CI: 2.45-4.82;
P<0.00001) increases in the risk of neutropenia when compared with wild type and
with at least one UGT1A1*1 allele, respectively. UGT1A1*1/*28 genotype had an OR
of 1.90 (95% CI: 1.44-2.51; P<0.00001) for an increased risk of neutropenia. A
twofold increase in risk of diarrhoea was associated with UGT1A1*28/*28 genotype
(OR=1.84, 95% CI: 1.24-2.72; P=0.002). In subgroup meta-analysis, the higher
incidence of diarrhoea in UGT1A1*28/*28 patients was limited to studies where
when IRI was given at higher doses (OR=2.37, 95% CI: 1.39-4.04; P=0.002) or
combined with 5-fluorouracil (FU or analogue) (OR=1.78, 95% CI: 1.16-2.75;
P=0.009). Genotyping of UGT1A1*28 polymorphism before treatment for CRC can
tailor IRI therapy and reduce the IRI-related toxicities. IRI-combined 5-FU (or
analogue) and a high-dose IRI therapy enhance IRI-induced diarrhoea among
patients bearing the UGT1A1*28 allele. Although the toxicity relationships were
much stronger with the UGT1A1*28 homozygous variant, associations were also
found with the UGT1A1*28 heterozygous variant. |
For which diseases members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics? | Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease (HD). | Various neurodegenerative diseases are associated with aberrant gene expression.
We recently identified a novel class of pimelic o-aminobenzamide histone
deacetylase (HDAC) inhibitors that show promise as therapeutics in the
neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease
(HD). Here, we describe the various techniques used in our laboratories to
dissect mechanisms of gene silencing in FRDA and HD, and to test our HDAC
inhibitors for their ability to reverse changes in gene expression in cellular
models. Numerous studies have pointed to histone deacetylase inhibitors as potential
therapeutics for various neurodegenerative diseases, and clinical trials with
several histone deacetylase inhibitors have been performed or are under way.
However, histone deacetylase inhibitors tested to date either are highly
cytotoxic or have very low specificities for different histone deacetylase
enzymes. The authors' laboratories have identified a novel class of histone
deacetylase inhibitors (2-aminobenzamides) that reverses
heterochromatin-mediated silencing of the frataxin (FXN) gene in Friedreich
ataxia. The authors have identified the histone deacetylase enzyme isotype
target of these compounds and present evidence that compounds that target this
enzyme selectively increase FXN expression from pathogenic alleles. Studies with
model compounds show that these histone deacetylase inhibitors increase FXN
messenger RNA levels in the brain in mouse models for Friedreich ataxia and
relieve neurological symptoms observed in mouse models and support the notion
that this class of molecules may serve as therapeutics for the human disease. Histone deacetylase (HDAC) inhibitors have received considerable attention as
potential therapeutics for a variety of cancers and neurological disorders.
Recent publications on a class of pimelic diphenylamide HDAC inhibitors have
highlighted their promise in the treatment of the neurodegenerative diseases
Friedreich's ataxia and Huntington's disease, based on efficacy in cell and
mouse models. These studies' authors have proposed that the unique action of
these compounds compared to hydroxamic acid-based HDAC inhibitors results from
their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3,
resulting in a distinctive pharmacological profile and reduced toxicity. Here,
we evaluate the HDAC subtype selectivity, cellular activity, absorption,
distribution, metabolism and excretion (ADME) properties, as well as the central
pharmacodynamic profile of one such compound, HDACi 4b, previously described to
show efficacy in vivo in the R6/2 mouse model of Huntington's disease. Based on
our data reported here, we conclude that while the in vitro selectivity and
binding mode are largely in agreement with previous reports, the physicochemical
properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b
render this compound suboptimal to investigate central Class I HDAC inhibition
in vivo in mouse per oral administration. A drug administration regimen using
HDACi 4b dissolved in drinking water was used in the previous proof of concept
study, casting doubt on the validation of CNS HDAC3 inhibition as a target for
the treatment of Huntington's disease. We highlight physicochemical stability
and metabolic issues with 4b that are likely intrinsic liabilities of the
benzamide chemotype in general. Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors
show promise as therapeutics for the neurodegenerative diseases Friedreich's
ataxia (FRDA) and Huntington's disease (HD). While it is clear that HDAC3 is one
of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of
other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial
effects of these compounds in FRDA and HD, and other HDAC interacting proteins
may be impacted by the compound. To this end, we synthesized activity-based
profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106),
and in the present study we used a quantitative proteomic method coupled with
multidimensional protein identification technology (MudPIT) to identify the
proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from
FRDA patient iPSC-derived neural stem cells, and then were reacted with control
and ABPP 106 probe. After reaction, the bound proteins were digested on the
beads, and the peptides were modified using stable isotope-labeled formaldehyde
to form dimethyl amine. The selectively bound proteins determined by mass
spectrometry were subjected to functional and pathway analysis. Our findings
suggest that the targets of compound 106 are involved not only in
transcriptional regulation but also in posttranscriptional processing of mRNA. |
Do U6-associated proteins Lsm4 and Lsm6 interact with SMN? | SMN interacts with at least two of the U6-associated Sm-like (Lsm) proteins, Lsm4 and Lsm6. | Arginine residues in RG-rich proteins are frequently dimethylated
posttranslationally by protein arginine methyltransferases (PRMTs). The most
common methylation pattern is asymmetrical dimethylation, a modification
important for protein shuttling and signal transduction. Symmetrically
dimethylated arginines (sDMA) have until now been confined to the myelin basic
protein MBP and the Sm proteins D1 and D3. We show here by mass spectrometry and
protein sequencing that also the human Sm protein B/B' and, for the first time,
one of the Sm-like proteins, LSm4, contain sDMA in vivo. The symmetrical
dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to
the Tudor domain of the "survival of motor neurons" protein (SMN): inhibition of
dimethylation by S-adenosylhomocysteine (SAH) abolished the binding of D1, D3,
B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine
dipeptides, but not asymmetrically modified or nonmodified peptides,
specifically inhibited the interaction of D1, D3, B/B', LSm4, and UsnRNPs with
SMN-Tudor. Recombit D1 and a synthetic peptide could be methylated in vitro
by both HeLa cytosolic S100 extract and nuclear extract; however, only the
cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying
PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a
prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. Our
demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in
tri-UsnRNP biogenesis and mRNA decay. Our findings also have interesting
implications for the understanding of the aetiology of spinal muscular atrophy
(SMA). Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and
this was quickly followed by the introduction of the LE cell test and indirect
immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical
laboratories. Recently, attention has turned to the identification of the
autoantigens that bind to cytoplasmic organelles such as the Golgi complex,
endosomes and other "cytoplasmic somes". Three endosome autoantigens include
early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170
(CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1
were seen in a variety of conditions but approximately 40% of the patients had a
neurological disease. Despite the prominence of lysosomes in cells and tissues,
reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the
cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex
include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130,
and golgin-67. More recently, there has been an interest in autoantibodies that
bind components of the "SMN complex" or the "assemblyosome". Arginine/glycine
(RG)-rich domains in components of the SMN complex interact with Sm, like-Sm
(LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are
antigen targets in a variety of diseases. More recently, components of a novel
cytoplasmic structure named GW bodies (GWBs) have been identified as targets of
human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding
protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn)
enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic
processing of mRNAs. Autoantibodies to the "cytoplasmic somes" are relatively
uncommon and serological tests to detect most of them are not widely available. The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP)
involved in histone messenger RNA (mRNA) 3' end formation has recently been
elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped
assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and
D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This
polypeptide composition and the unusual structure of Lsm11, which plays a role
in histone RNA processing, represent new themes in the biology of Sm/Lsm
proteins. Moreover this structure has important consequences for snRNP assembly
that is mediated by two complexes containing the PRMT5 methyltransferase and the
SMN (survival of motor neurons) protein, respectively. Finally, the ability to
alter this polypeptide composition by a small mutation in U7 snRNA forms the
basis for using modified U7 snRNA derivatives to alter specific pre-mRNA
splicing events, thereby opening up a new way for antisense gene therapy. Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle
involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly
imported Sm-class snRNPs are thought to traffic through CBs before proceeding to
their final nuclear destinations. Loss of coilin function in mice leads to
significant viability and fertility problems. Coilin interacts directly with the
spinal muscular atrophy (SMA) protein via dimethylarginine residues in its
C-terminal domain. Although coilin hypomethylation results in delocalization of
survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain
within these structures. Thus, CBs appear to be involved in snRNP maturation,
but factors that tether snRNPs to CBs have not been described. In this report,
we demonstrate that the coilin C-terminal domain binds directly to various Sm
and Lsm proteins via their Sm motifs. We show that the region of coilin
responsible for this binding activity is separable from that which binds to SMN.
Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal
domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7
snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as
well as with intact snRNPs, indicates that coilin and CBs may facilitate the
modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the
reclamation of unassembled snRNP components. Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U
snRNPs) is a process facilitated by the macromolecular survival of motor neuron
(SMN) complex. This entity promotes the binding of a set of factors, termed
LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine
factors, including the SMN protein, the product of the spinal muscular atrophy
(SMA) disease gene, Gemins 2-8 and unrip have been identified as the major
components of the SMN complex. So far, however, only little is known about the
architecture of this complex and the contribution of individual components to
its function. Here, we present a comprehensive interaction map of all core
components of the SMN complex based upon in vivo and in vitro methods. Our
studies reveal a modular composition of the SMN complex with the three proteins
SMN, Gemin8, and Gemin7 in its center. Onto this central building block the
other components are bound via multiple interactions. Furthermore, by employing
a novel assay, we were able to reconstitute the SMN complex from individual
components and confirm the interaction map. Interestingly, SMN protein carrying
an SMA-causing mutation was severely impaired in formation of the SMN complex.
Finally, we show that the peripheral component Gemin5 contributes an essential
activity to the SMN complex, most likely the transfer of Sm proteins onto the U
snRNA. Collectively, the data presented here provide a basis for the detailed
mechanistic and structural analysis of the assembly machinery of U snRNPs. Reduced levels of survival of motoneuron (SMN) protein lead to spinal muscular
atrophy, but it is still unknown how SMN protects motoneurons in the spinal cord
against degeneration. In the nucleus, SMN is associated with two types of
nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of
fibroblast growth factor-2 (FGF-2(23)) is a nuclear protein that binds to SMN
and destabilizes the SMN-Gemin2 complex. In the present study, we show that
FGF-2(23) depletes SMN from CBs without affecting their general structure. FRAP
analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs
remained mobile and allowed quantification of fast, slow and immobile nuclear
SMN populations. The potential for SMN release was confirmed by in vivo
photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN
that could have a specialized function in CBs. FGF-2(23) accelerated SMN release
from CBs, accompanied by a conversion of immobile SMN into a mobile population.
Furthermore, FGF-2(23) caused snRNP accumulation in CBs. We propose a model in
which Cajal bodies store immobile SMN that can be mobilized by its nuclear
interaction partner FGF-2(23), leading to U4 snRNP accumulation in CBs,
indicating a role for immobile SMN in tri-snRNP assembly. |
List anti-amyloid-beta monoclonal antibodies that have been investigated in clinical trials for treatment of Alzheimer disease. | Ponezumab, solanezumab and bapineuzumab are humanized antiamyloid beta (Aβ) monoclonal antibodies that have been designed for treatment of Alzheimer disease. | The deposition of amyloid beta (Abeta) protein is a key pathological feature in
Alzheimer's disease (AD). In murine models of AD, both active and passive
immunization against Abeta induce a marked reduction in amyloid brain burden and
an improvement in cognitive functions. Preliminary results of a prematurely
terminated clinical trial where AD patients were actively vaccinated with
aggregated Abeta bear resemblance to those documented in murine models. Passive
immunization of AD patients with anti-Abeta antibodies, in particular human
antibodies, is a strategy that provides a more cautious management and control
of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG
autoimmune antibodies. Hence antigen-committed human B-cells are easily
immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines.
Two anti-Abeta human monoclonal antibodies which we recently prepared bind to
the N-terminus of Abeta peptide and were shown to stain amyloid plaques in
non-fixed brain sections from an AD patient. It is anticipated that specifically
selected anti-Abeta human monoclonal antibodies could reduce and inhibit
deposits of amyloid in brain while avoiding the cognitive decline that
characterizes AD. In the future, this type of antibody may prove to be a
promising immune therapy for the disease. Ya que la población mundial sigue envejeciendo, la Enfermedad de Alzheimer
presenta una crisis inminente para la salud pública, que si se descuida,
amenazará con sobrecargar los sistemas de atención de salud en el mundo
desarrollado. Para abordar significativamente el síntoma más catastrófico y
devastador de la Enfermedad de Alzheimer (EA), la dementia, debemos ser capaces
de detectar la enfermedad antes de que aparezcan los síntomas clínicos, y
ofrecer a los pacientes tratamientos preventivos que bloqueen o retrasen
significativamente la progresión de la enfermedad. Esta revisión resume varios
de los métodos más prometedores de detección precoz para la EA y el deterioro
cognitivo leve (DCL) que podrían ser utilizados para identificar a los pacientes
con alto riesgo de desarrollar la enfermedad y para monitorear la progresión de
ésta y la respuesta a tratamientos en investigatión. Además, se destacan algunos
de los programas de tratamiento en investigatión que podrían llegar a constituir
terapias modificadoras de la enfermedad, que retrasen significativamente el
desarrollo de la dementia. Estos potentiates tratamientos están dirigidos a muy
diversas vías, y un día podrán ser administrados en combinación para aumentar la
eficatia y prévenir el deterioro cognitivo en patientes con EA. Aunque todavía
se enfrentan numerosos desafíos, los investigadores de la EA han realizado
grandes progresos para la comprensión de los mecanismes de la enfermedad. Como
se ha observado en el tratamiento de la enfermedad cardíaca, incluso modestos
tratamientos preventives pueden tener un gran impacto en la evolutión clínica y
reducir drásticamente la prevalentia de la enfermedad en un subgrupo de la
población. Por lo tanto, hay esperanzas en que el desarrollo de tratamientos
profilácticos combinado con una mejoría en los métodos de detectión precoz,
proveerá un dramático alivio para millones de individuos que están envejeciendo
amenazados por el espectro de la Enfermedad de Alzheimer. BACKGROUND: Bapineuzumab, a humanized anti-amyloid-beta (Abeta) monoclonal
antibody for the potential treatment of Alzheimer disease (AD), was evaluated in
a multiple ascending dose, safety, and efficacy study in mild to moderate AD.
METHODS: The study enrolled 234 patients, randomly assigned to IV bapineuzumab
or placebo in 4 dose cohorts (0.15, 0.5, 1.0, or 2.0 mg/kg). Patients received 6
infusions, 13 weeks apart, with final assessments at week 78. The prespecified
primary efficacy analysis in the modified intent-to-treat population assumed
linear decline and compared treatment differences within dose cohorts on the
Alzheimer's Disease Assessment Scale-Cognitive and Disability Assessment for
Dementia. Exploratory analyses combined dose cohorts and did not assume a
specific pattern of decline.
RESULTS: No significant differences were found in the primary efficacy analysis.
Exploratory analyses showed potential treatment differences (p < 0.05,
unadjusted for multiple comparisons) on cognitive and functional endpoints in
study "completers" and APOE epsilon4 noncarriers. Reversible vasogenic edema,
detected on brain MRI in 12/124 (9.7%) bapineuzumab-treated patients, was more
frequent in higher dose groups and APOE epsilon4 carriers. Six vasogenic edema
patients were asymptomatic; 6 experienced transient symptoms.
CONCLUSIONS: Primary efficacy outcomes in this phase 2 trial were not
significant. Potential treatment differences in the exploratory analyses support
further investigation of bapineuzumab in phase 3 with special attention to APOE
epsilon4 carrier status.
CLASSIFICATION OF EVIDENCE: Due to varying doses and a lack of statistical
precision, this Class II ascending dose trial provides insufficient evidence to
support or refute a benefit of bapineuzumab. BACKGROUND: Carbon-11-labelled Pittsburgh compound B ((11)C-PiB) PET is a marker
of cortical fibrillar amyloid-beta load in vivo. We used (11)C-PiB PET to
investigate whether bapineuzumab, a humanised anti-amyloid-beta monoclonal
antibody, would reduce cortical fibrillar amyloid-beta load in patients with
Alzheimer's disease.
METHODS: Patients with mild-to-moderate Alzheimer's disease were randomly
assigned to receive intravenous bapineuzumab or placebo in a ratio of seven to
three in three ascending dose groups (0.5, 1.0, or 2.0 mg/kg). Each dose group
was enrolled after safety review of the previous group. Randomisation was by
interactive voice response system; masking was achieved with numbered kit
allocation. Patients, investigators, study site personnel, sponsor staff, and
carers were masked to treatment. Patients received up to six infusions, 13 weeks
apart, and had (11)C-PiB PET scans at baseline and at weeks 20, 45, and 78. The
primary outcome was the difference between the pooled bapineuzumab group and the
pooled placebo group in mean change from screening to week 78 in (11)C-PiB
cortical to cerebellar retention ratio averaged across six cortical regions of
interest. Analysis was by modified intention to treat. This study is registered
with EudraCT, number 2004-004120-12; ISRCTN17517446.
FINDINGS: 28 patients were assigned to bapineuzumab (n=20) or placebo (n=8). 19
patients in the bapineuzumab group and seven in the placebo group were included
in the modified intention-to-treat analysis. Estimated mean (11)C-PiB retention
ratio change from baseline to week 78 was -0.09 (95% CI -0.16 to -0.02; p=0.014)
in the bapineuzumab group and 0.15 (95% CI 0.02 to 0.28; p=0.022) in the placebo
group. Estimated mean difference in (11)C-PiB retention ratio change from
baseline to week 78 between the bapineuzumab group and the placebo group was
-0.24 (95% CI -0.39 to -0.09; p=0.003). Differences between the bapineuzumab
group and the placebo group in the individual regions of interest were similar
to the overall mean difference. Adverse events were typically mild to moderate
in severity and transient. Two patients in the 2.0 mg/kg bapineuzumab group had
transient cerebral vasogenic oedema.
INTERPRETATION: Treatment with bapineuzumab for 78 weeks reduced cortical
(11)C-PiB retention compared with both baseline and placebo. (11)C-PiB PET seems
to be useful in assessing the effects of potential Alzheimer's disease
treatments on cortical fibrillar amyloid-beta load in vivo.
FUNDING: Elan Pharmaceuticals and Wyeth Research. Pre-clinical and clinical data suggest that the development of a safe and
effective anti-amyloid-beta (Abeta) immunotherapy for Alzheimer's disease (AD)
will require therapeutic levels of anti-Abeta antibodies, while avoiding
proinflammatory adjuvants and autoreactive T cells which may increase the
incidence of adverse events in the elderly population targeted to receive
immunotherapy. The first active immunization clinical trial with AN1792 in AD
patients was halted when a subset of patients developed meningoencephalitis. The
first passive immunotherapy trial with bapineuzumab, a humanized monoclonal
antibody against the end terminus of Abeta, also encountered some dose dependent
adverse events during the Phase II portion of the study, vasogenic edema in 12
cases, which were significantly over represented in ApoE4 carriers. The proposed
remedy is to treat future patients with lower doses, particularly in the ApoE4
carriers. Currently there are at least five ongoing anti-Abeta immunotherapy
clinical trials. Three of the clinical trials use humanized monoclonal
antibodies, which are expensive and require repeated dosing to maintain
therapeutic levels of the antibodies in the patient. However in the event of an
adverse response to the passive therapy antibody delivery can simply be halted,
which may provide a resolution to the problem. Because at this point we cannot
readily identify individuals in the preclinical or prodromal stages of AD
pathogenesis, passive immunotherapy is reserved for those that already have
clinical symptoms. Unfortunately those individuals have by that point
accumulated substantial neuropathology in affected regions of the brain.
Moreover, if Abeta pathology drives tau pathology as reported in several
transgenic animal models, and once established if tau pathology can become self
propagating, then early intervention with anti-Abeta immunotherapy may be
critical for favorable clinical outcomes. On the other hand, active immunization
has several significant advantages, including lower cost and the typical
immunization protocol should be much less intrusive to the patient relative to
passive therapy, in the advent of Abeta-antibody immune complex-induced adverse
events the patients will have to receive immuno-supperssive therapy for an
extended period until the anti Abeta antibody levels drop naturally as the
effects of the vaccine decays over time. Obviously, improvements in vaccine
design are needed to improve both the safety, as well as the efficacy of
anti-Abeta immunotherapy. The focus of this review is on the advantages of DNA
vaccination for anti-Abeta immunotherapy, and the major hurdles, such as
immunosenescence, selection of appropriate molecular adjuvants, universal T cell
epitopes, and possibly a polyepitope design based on utilizing existing memory T
cells in the general population that were generated in response to childhood or
seasonal vaccines, as well as various infections. Ultimately, we believe that
the further refinement of our AD DNA epitope vaccines, possibly combined with a
prime boost regime will facilitate translation to human clinical trials in
either very early AD, or preferably in preclinical stage individuals identified
by validated AD biomarkers. Anti-amyloid-beta immunization leads to amyloid clearance in patients with
Alzheimer's disease, but the effect of vaccination on amyloid-beta-induced
neuronal pathology has not been quantitatively examined. The objectives of this
study were to address the effects of anti-amyloid-beta active immunization on
neurite trajectories and the pathological hallmarks of Alzheimer's disease in
the human hippocampus. Hippocampal sections from five patients with Alzheimer's
disease enrolled in the AN1792 Phase 2a trial were compared with those from 13
non-immunized Braak-stage and age-matched patients with Alzheimer's disease, and
eight age-matched non-demented controls. Analyses included neurite curvature
ratio as a quantitative measure of neuritic abnormalities, amyloid and tau
loads, and a quantitative characterization of plaque-associated neuritic
dystrophy and astrocytosis. Amyloid load and density of dense-core plaques were
decreased in the immunized group compared to non-immunized patients (P < 0.01
and P < 0.001, respectively). The curvature ratio in non-immunized patients with
Alzheimer's disease was elevated compared to non-demented controls (P < 0.0001).
In immunized patients, however, the curvature ratio was normalized when compared
to non-immunized patients (P < 0.0001), and not different from non-demented
controls. In the non-immunized patients, neurites close to dense-core plaques
(within 50 microm) were more abnormal than those far from plaques (i.e. beyond
50 microm) (P < 0.0001). By contrast, in the immunized group neurites close to
and far from the remaining dense-core plaques did not differ, and both were
straighter compared to the non-immunized patients (P < 0.0001). Compared to
non-immunized patients, dense-core plaques remaining after immunization had
similar degree of astrocytosis (P = 0.6060), more embedded dystrophic neurites
(P < 0.0001) and were more likely to have mitochondrial accumulation (P <
0.001). In addition, there was a significant decrease in the density of paired
helical filament-1-positive neurons in the immunized group as compared to the
non-immunized (P < 0.05), but not in the density of Alz50 or thioflavin-S
positive tangles, suggesting a modest effect of anti-amyloid-beta immunization
on tangle pathology. Clearance of amyloid plaques upon immunization with AN1792
effectively improves a morphological measure of neurite abnormality in the
hippocampus. This improvement is not just attributable to the decrease in plaque
load, but also occurs within the halo of the remaining dense-core plaques.
However, these remaining plaques still retain some of their toxic potential.
Anti-amyloid-beta immunization might also ameliorate the hippocampal tau
pathology through a decrease in tau phosphorylation. These data agree with
preclinical animal studies and further demonstrate that human anti-amyloid-beta
immunization does not merely clear amyloid from the Alzheimer's disease brain,
but reduces some of the neuronal alterations that characterize Alzheimer's
disease. OBJECTIVES: Active and passive immunization strategies have been suggested as
possible options for the treatment of Alzheimer disease (AD). LY2062430
(solanezumab) is a humanized monoclonal antibody being studied as a putative
disease-modifying treatment of AD.
METHODS: Patients with mild to moderate AD were screened and selected for
inclusion. Initial screening was performed for 54 subjects, and 29 of these
underwent additional screening; after this second screening, a total of 19
subjects were included. Single doses of solanezumab using 0.5, 1.5, 4.0, and
10.0 mg/kg were administered. Safety assessments included gadolinium-enhanced
magnetic resoce imaging of the brain and cerebrospinal fluid (CSF) analyses
at baseline and 21 days after dosing. Plasma and CSF concentrations of
solanezumab and amyloid beta (Abeta) and cognitive evaluations were obtained.
RESULTS: Administration of solanezumab was generally well tolerated except that
mild self-limited symptoms consistent with infusion reactions occurred for 2 of
4 subjects given 10 mg/kg. No evidence of meningoencephalitis, microhemorrhage,
or vasogenic edema was present based on magnetic resoce image and CSF
analyses. A substantial dose-dependent increase in total (bound plus unbound)
Abeta was demonstrated in plasma; CSF total Abeta also increased. No changes in
cognitive scores occurred.
CONCLUSIONS: A single dose of solanezumab was generally well tolerated, although
infusion reactions similar to those seen with administration of other proteins
may occur with higher doses. A dose-dependent change in plasma and CSF Abeta was
observed, although changes in cognitive scores were not noted. Further studies
of solanezumab for the treatment of AD are warranted. The safety, tolerability, and pharmacokinetics (PKs) of bapineuzumab (AAB-001),
a humanized monoclonal antibody to amyloid beta, were evaluated in patients with
mild-to-moderate Alzheimer disease in a phase 1, randomized, third-party
unblinded, placebo-controlled, single ascending dose trial. Thirty patients
received bapineuzumab infusion of 0.5, 1.5, or 5 mg/kg or placebo (6 active, 2
placebo for 0.5 and 1.5-mg/kg cohorts; 10 active, 4 placebo for 5.0-mg/kg
cohort). Three patients in the highest dose cohort (5.0 mg/kg) developed
magnetic resoce imaging abnormalities consistent with vasogenic edema,
predomitly high signal abnormalities on fluid-attenuated inversion recovery
sequences, all of which resolved over time. Plasma amyloid beta was elevated
from baseline, peaking approximately 24 hours after infusion. PK analysis
demonstrated a half-life of 21 to 26 days, supporting a 13-week dosing interval
for bapineuzumab. This small, single-dose study demonstrated the safety profile
and PK characteristics of bapineuzumab and was used to design later safety and
efficacy trials. The field of Alzheimer's disease (AD) research eagerly awaits the results of a
large number of Phase III clinical trials that are underway to investigate the
effectiveness of anti-amyloid-β (Aβ) immunotherapy for AD. In this case report,
we review the pertinent clinical history, examine the neuropathology, and
characterize the Aβ profile of an AD patient who received bapineuzumab
immunotherapy. The patient received four bapineuzumab infusions over a 39 week
period. During the course of this treatment, there was no remarkable change in
cognitive impairment as determined by MMSE scores. Forty-eight days after the
fourth bapineuzumab infusion was given, MRI revealed that the patient had
developed lacunar infarcts and possible vasogenic edema, probably related to
immunotherapy, but a subsequent MRI scan 38 days later demonstrated resolution
of vasogenic edema. The patient expired due to acute congestive heart failure
complicated by progressive AD and cerebrovascular accident 378 days after the
first bapineuzumab infusion and 107 days after the end of therapy.
Neuropathological and biochemical analysis did not produce evidence of lasting
plaque regression or clearance of Aβ due to immunotherapy. The Aβ species
profile of this case was compared with non-immunized AD cases and non-demented
controls and found to be similar to non-immunized AD cases. SELDI-TOF mass
spectrometric analysis revealed the presence of full-length Aβ₁₋₄₂ and truncated
Aβ peptides demonstrating species with and without bapineuzumab specific
epitopes. These results suggest that, in this particular case, bapineuzumab
immunotherapy neither resulted in detectable clearance of amyloid plaques nor
prevented further cognitive impairment. INTRODUCTION: Alzheimer's disease (AD) is a debilitating neurodegenerative
illness affecting over 35 million people worldwide. Solanezumab is a monoclonal
antibody that binds to β-amyloid (Aβ), a protein that plays a key role in the
pathogenesis of AD. The drug is currently being investigated in Phase III trials
as a disease-modifying treatment for AD.
AREAS COVERED: This paper reviews literature on solanezumab that is available in
PubMed from 2008 to 2010, other treatment trials in clinicaltrials.gov and
published abstracts from conferences. The article also provides a discussion of
the early trials of AN1792 and an overview of the immunotherapies currently in
development. The authors provide the reader with a critical appraisal of the
to-date clinical trial data on solanezumab and its implications for the broader
field of immunotherapies for AD.
EXPERT OPINION: Solanezumab can neutralize soluble Aβ peptides, which may
represent the more neurotoxic of the Aβ species. Phase II findings support the
compound's safety, which has been a concern for some Aβ immunotherapies.
Cerebrospinal and plasma biomarker changes with solanezumab treatment are
encouraging. Results of the ongoing Phase III trials will be instrumental in
determining the drug's clinical significance. BACKGROUND: Cerebral vasogenic edema (VE) has been reported to occur during
antiamyloid immunotherapy. VE may be associated with central nervous system
pathology with blood-brain barrier disruptions; however, less is known about the
prevalence of naturally occurring VE in patients with Alzheimer's disease (AD).
METHODS: Fluid-attenuated inversion recovery imaging sequences were obtained
from four ongoing multicenter, randomized, double-blind, placebo-controlled,
phase 3 trials in patients with mild-to-moderate AD. The first set of baseline
scans was from patients in volumetric magnetic resoce imaging addenda in the
Interrupting Alzheimer's Dementia by EvaluatiNg Treatment of Amyloid PaThologY
(IDENTITY) studies examining semagacestat, a γ-secretase inhibitor (cohort 1, n
= 621). The second set of baseline scans was from the EXPanding alzhEimer's
Disease InvestigaTIONs (EXPEDITION) studies examining solanezumab, an anti-Aβ
monoclonal antibody (cohort 2, n = 2141). Readers were blinded to
patient-identifying information and future treatment. A third set of baseline
scans was from the first 700 patients who underwent protocol-specified magnetic
resoce imaging before randomization in the EXPEDITION studies (cohort 3). The
analysis used three neuroradiologists: two performed independent primary
interpretations and the third was the adjudicator. Readers were blinded to
patient information, treatment, protocol, and time point.
RESULTS: Four cases of asymptomatic VE were detected at baseline/screening. Two
VE cases were due to underlying extra-axial mass lesions. The third VE case was
associated with numerous microhemorrhages in keeping with cerebral amyloid
angiopathy-related inflammation or Aβ-related angiitis. The final VE case
demonstrated localized sulcal fluid-attenuated inversion recovery imaging
hyperintensity. No VE was detected in cohort 3 by readers blinded to patient
baseline status.
CONCLUSIONS: VE seems to be rare at baseline in patients with AD in clinical
trials, 2 of 2,762 associated with AD. Additional cohorts should be evaluated to
support these findings. As the societal and economic burdens of Alzheimer's disease (AD) continue to
mount, so does the need for therapies that slow the progression of the illness.
Beta amyloid has long been recognized as the pathologic hallmark of AD, and the
past decade has seen significant progress in the development of various
immunotherapeutic approaches targeting beta amyloid. This paper reviews active
and passive approaches aimed at beta amyloid, with a focus on clinical trial
data. The exact mechanisms leading to Alzheimer's disease (AD) are largely unknown,
limiting the identification of effective disease-modifying therapies. The two
principal neuropathological hallmarks of AD are extracellular β-amyloid (Aβ),
peptide deposition (senile plaques) and intracellular neurofibrillary tangles
containing hyperphosphorylated tau protein. During the last decade, most of the
efforts of the pharmaceutical industry were directed against the production and
accumulation of Aβ. The most innovative of the pharmacological approaches was
the stimulation of Aβ clearance from the brain of AD patients via the
administration of Aβ antigens (active vaccination) or anti-Aβ antibodies
(passive vaccination). Several active and passive anti-Aβ vaccines are under
clinical investigation. Unfortunately, the first active vaccine (AN1792,
consisting of preaggregate Aβ and an immune adjuvant, QS-21) was abandoned
because it caused meningoencephalitis in approximately 6% of treated patients.
Anti-Aβ monoclonal antibodies (bapineuzumab and solanezumab) are now being
developed. The clinical results of the initial studies with bapineuzumab were
equivocal in terms of cognitive benefit. The occurrence of vasogenic edema after
bapineuzumab, and more rarely brain microhemorrhages (especially in Apo E ε4
carriers), has raised concerns on the safety of these antibodies directed
against the N-terminus of the Aβ peptide. Solanezumab, a humanized anti-Aβ
monoclonal antibody directed against the midregion of the Aβ peptide, was shown
to neutralize soluble Aβ species. Phase II studies showed a good safety profile
of solanezumab, while studies on cerebrospinal and plasma biomarkers documented
good signals of pharmacodynamic activity. Although some studies suggested that
active immunization may be effective against tau in animal models of AD, very
few studies regarding passive immunization against tau protein are currently
available. The results of the large, ongoing Phase III trials with bapineuzumab
and solanezumab will tell us if monoclonal anti-Aβ antibodies may slow down the
rate of deterioration of AD. Based on the new diagnostic criteria of AD and on
recent major failures of anti-Aβ drugs in mild-to-moderate AD patients, one
could argue that clinical trials on potential disease-modifying drugs, including
immunological approaches, should be performed in the early stages of AD. BACKGROUND: Given the slow and variable clinical course of Alzheimer disease,
very large and extended clinical trials are needed to identify a beneficial
clinical effect of disease-modifying treatments. Therefore, biomarkers are
essential to prove that an anti-β-amyloid (Aβ) drug candidate affects both Aβ
metabolism and plaque load as well as downstream pathogenic mechanisms.
OBJECTIVE: To evaluate the effect of the anti-Aβ monoclonal antibody
bapineuzumab on cerebrospinal fluid (CSF) biomarkers reflecting Aβ homeostasis,
neuronal degeneration, and tau-related pathology in patients with Alzheimer
disease.
DESIGN: Two phase 2, multicenter, randomized, double-blind, placebo-controlled
clinical trials of 12-month duration.
SETTING: Academic centers in the United States (Study 201) and England and
Finland (Study 202).
PATIENTS: Forty-six patients with mild to moderate Alzheimer disease.
INTERVENTIONS: Patients received either placebo (n = 19) or bapineuzumab (n =
27) in 3 or 4 ascending dose groups.
MAIN OUTCOME MEASURES: Changes between end of study and baseline in the
exploratory CSF biomarkers Aβ1-42, AβX-42, AβX-40; total tau (T-tau); and
phosphorylated tau (P-tau).
RESULTS: Within the bapineuzumab group, a decrease at end of study compared with
baseline was found both for CSF T-tau (-72.3 pg/mL) and P-tau (-9.9 pg/mL). When
comparing the treatment and placebo groups, this difference was statistically
significant for P-tau (P = .03), while a similar trend for a decrease was found
for T-tau (P = .09). No clear-cut differences were observed for CSF Aβ.
CONCLUSIONS: To our knowledge, this study is the first to show that passive Aβ
immunotherapy with bapineuzumab results in decreases in CSF T-tau and P-tau,
which may indicate downstream effects on the degenerative process. Cerebrospinal
fluid biomarkers may be useful to monitor the effects of novel disease-modifying
anti-Aβ drugs in clinical trials. TRIAL REGISTRATIONS clinicaltrials.gov
Identifier: NCT00112073, EudraCT Identifier: 2004-004120-12, and isrctn.org
Identifier: ISRCTN17517446. OBJECTIVE: Ponezumab (PF-04360365) is a humanized anti-amyloid beta (Aβ)
monoclonal antibody designed for treatment of Alzheimer disease (AD). A single
2-hour intravenous infusion of 0.1 to 10 mg/kg was previously shown to be safe
and well tolerated in subjects with mild to moderate AD, with measurable effects
on plasma and cerebrospinal fluid Aβ. This phase I, dose-escalation, open-label
study evaluated the safety, pharmacokinetics, and pharmacodynamics of a single
10-minute intravenous infusion.
METHODS: Subjects with mild to moderate AD received ponezumab 1 mg/kg (n = 3), 3
mg/kg (n = 3), 5 mg/kg (n = 4), or 10 mg/kg (n = 5). They were followed up as
outpatients for 6 months.
RESULTS: All subjects completed the trial. Ponezumab was safe and well tolerated
with no deaths, withdrawals, or drug-related moderate, severe, or serious
adverse events. Mild drug-related adverse events included headache (3 patients)
and lethargy and hypoesthesia (both in 1 patient). No infusion reactions,
clinically meaningful laboratory abnormalities, vital sign changes,
electrocardiographic changes, or antidrug antibodies were detected. There was no
evidence of brain microhemorrhage, vasogenic edema, encephalitis, or other
imaging abnormality. Cognitive function showed no treatment-related trends.
Ponezumab displayed approximately dose-proportional increases in plasma
exposure. Steady-state volume of distribution was 113 to 172 mL/kg, clearance
was 2.7 to 3.0 mL/d/kg, and terminal half-life was 35 to 52 days. Plasma maximum
observed concentration and the area under the plasma concentration-time profile
from time 0 extrapolated to infinite time of Aβ(1-x) and Aβ(1-40) increased
dose-dependently.
CONCLUSIONS: Administration of ponezumab as a 10-minute infusion was safe and
well tolerated and produced effects on plasma Aβ species comparable with a
2-hour infusion. Shorter infusions may provide more flexibility, comfort, and
convenience for patients and caregivers. OBJECTIVES: Ponezumab is a humanized antiamyloid beta (Aβ) monoclonal antibody
designed to treat Alzheimer disease (AD).
METHODS: This randomized, double-blind, single-dose-escalation study evaluated
the safety, pharmacokinetics, and pharmacodynamics of 0.1, 0.3, 1, 3, and 10
mg/kg ponezumab (n = 4, 4, 4, 6, and 8, respectively) versus placebo (n = 11)
after a 2-hour intravenous infusion in subjects with mild-to-moderate AD.
Cerebrospinal fluid (CSF) samples were obtained from the 1- and 10-mg/kg groups
at baseline and at day 29. The subjects were followed for 1 year.
RESULTS: All subjects completed the trial. Ponezumab was well tolerated with no
drug-attributed serious adverse events. The most common adverse events were
upper respiratory tract infection, headache, and back pain, all mild to
moderate. One subject (10 mg/kg) experienced a mild hypersensitivity reaction.
Another subject (0.1 mg/kg) demonstrated slight enlargement of a preexisting
midbrain lesion. Electrocardiography and laboratory values (including CSF) were
unremarkable. No evidence of new microhemorrhage, vasogenic edema, or
meningoencephalitis was noted. Plasma maximum observed concentration increased
approximately dose proportionally, and the area under the plasma
concentration-time profile from time zero extrapolated to infinite time
(AUC(inf)) increased slightly more than dose proportionally. Mean terminal
half-life was approximately 6 weeks. Two subjects (10 mg/kg) had measurable CSF
ponezumab concentrations (~0.5% of plasma values) at day 29. Plasma Aβ(1-x) and
Aβ(1-40) increased dose dependently, and mean CSF Aβ(1-x) increased 38% from
baseline with 10 mg/kg (P = 0.002 vs placebo).
CONCLUSIONS: A 2-hour infusion of 0.1 to 10 mg/kg ponezumab was well tolerated
in subjects with mild-to-moderate AD. Plasma pharmacokinetic profile was
approximately linear. Plasma Aβ increased with dose, and CSF Aβ increased at the
highest dose, suggesting that intravenous ponezumab alters central Aβ levels. Anti-amyloid beta (Aβ) immunotherapy provides potential benefits in Alzheimer's
disease patients. Nevertheless, strategies based on Aβ1-42 peptide induced
encephalomyelitis and possible microhemorrhages. These outcomes were not
expected from studies performed in rodents. It is critical to determine if other
animal models better predict side effects of immunotherapies. Mouse lemur
primates can develop amyloidosis with aging. Here we used old lemurs to study
immunotherapy based on Aβ1-42 or Aβ-derivative (K6Aβ1-30). We followed anti-Aβ40
immunoglobulin G and M responses and Aβ levels in plasma. In vivo magnetic
resoce imaging and histology were used to evaluate amyloidosis,
neuroinflammation, vasogenic edema, microhemorrhages, and brain iron deposits.
The animals responded mainly to the Aβ1-42 immunogen. This treatment induced
immune response and increased Aβ levels in plasma and also microhemorrhages and
iron deposits in the choroid plexus. A complementary study of untreated lemurs
showed iron accumulation in the choroid plexus with normal aging. Worsening of
iron accumulation is thus a potential side effect of Aβ-immunization at
prodromal stages of Alzheimer's disease, and should be monitored in clinical
trials. |
Does MVIIA and MVIIC bind to the same calcium channel? | No, the omega-conotoxin MVIIC blocks P/Q-type calcium channels with high affinity and N-type calcium channels with low affinity, while the highly homologous omega-conotoxin MVIIA blocks only N-type calcium channels. | High-threshold voltage-sensitive calcium channels of the N-type, L-type, and
P-type have been distinguished in the mammalian CNS predomitly on the basis
of their sensitivity to selective antagonists. Matching them with genes
identified by molecular cloning is an ongoing undertaking. Whereas L-type
channels are characterized by their sensitivity to dihydropyridines and P-type
channels by sensitivity to the funnel-web spider toxin AgaIVA, the N-type
channel has been shown to be recognized by the omega-conopeptides GVIA and
MVIIA. Recently, two new members of the family of omega-conopeptides--MVIIC from
the marine snail Conus magus and SVIB from Conus striatus--have been described.
Binding and electrophysiological data suggest that these two peptides, in
addition to interacting with N-type calcium channels, interact with a widely
distributed receptor in neuronal membranes that is distinct from N-type
channels. In this report we demonstrate through biochemical and pharmacological
differentiation at individual receptor polypeptide resolution, by affinity
cross-linking, SDS-PAGE, and autoradiography, that SNX-230 (synthetic MVIIC)
binds with high affinity to a calcium channel alpha 1 subunit distinct from the
high-affinity alpha 1 target of SNX-111 (synthetic MVIIA). SNX-183 (synthetic
SVIB) interacts with both alpha 1 subunits with lower affinity. Whereas the
alpha 1 subunit recognized with high affinity by MVIIA corresponds to the N-type
channel, the other represents a novel calcium channel distinct from N-, L-, and
perhaps P-type channels. This study investigated the behavioural and anticonvulsant effects of
voltage-sensitive calcium channel blockers in DBA/2 mice. Omega-Conotoxin MVIIC
(0.1, 0.3 micrograms ICV/mouse) and omega-agatoxin IVA (0.1, 0.3, 1 micrograms
ICV), which act predomitly at P- and/or Q-type calcium channels, prevented
clonic and tonic sound-induced seizures in this animal model of reflex epilepsy
(ED50 values with 95% confidence limits for protection against clonic
sound-induced seizures were 0.09 (0.04-0.36) micrograms ICV and 0.09 (0.05-0.15)
micrograms ICV respectively and against tonic seizures 0.07 (0.03-0.16)
micrograms ICV and 0.08 (0.04-0.13) micrograms ICV, respectively). The N-type
calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA were
also tested in this model. Omega-Conotoxin GVIA was anticonvulsant in DBA/2
mice, but only at high doses (3 micrograms ICV prevented tonic seizures in 60%
of the animals; 10 micrograms ICV prevented clonic seizures in 60% and tonic
seizures in 90% of the animals), whereas omega-conotoxin MVIIA did not inhibit
sound-induced seizures in doses up to 10 micrograms ICV. Both omega-conotoxin
GVIA and omega-conotoxin MVIIA induced an intense shaking syndrome in doses as
low as 0.1 microgram ICV, whereas omega-conotoxin MVIIC and omega-agatoxin IVA
did not produce shaking at any of the doses examined. Finally, omega-conotoxin
GI (0.01-1 microgram ICV) and alpha-conotoxin SI (0.3-30 micrograms ICV), which
both act at acetylcholine nicotinic receptors, were not anticonvulsant and did
not induce shaking in DBA/2 mice. These results confirm that blockers of N- and
P-/Q-type calcium channels produce different behavioural responses in animals.
The anticonvulsant effects of omega-conotoxin MVIIC and omega-agatoxin IVA in
DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel
blockers inhibit the release of excitatory amino acids and are worthy of further
exploration. Electrically-induced twitch responses of the prostatic segment of vas deferens
(0.1 Hz, 65 V, 1 ms) are mainly due to the transient presynaptic release of ATP,
which acts postsynaptically on non-adrenergic receptors to contract smooth
muscle cells. These responses were fully blocked by omolar concentrations of
the omega-conotoxins GVIA, MVIIA, and MVIIC, most likely by inhibiting Ca2+
entry through presynaptic N-type Ca2+ channels controlling the release of ATP.
Repeated washout of the toxins allowed the recovery of contractions, except for
omega-conotoxin GVIA, whose inhibitory effects remained unchanged for at least
60 min. In addition, micromolar concentrations of omega-conotoxin MVIIC were
unable to protect against the irreversible inhibition of twitch contractions
induced by omolar concentrations of omega-conotoxin GVIA. At low
extracellular Ca2+ concentrations (1.5 mM), 20 nM of omega-conotoxin GVIA or
MVIIA inhibited completely the twitch contractions in about 10 min. In 5 mM Ca2+
the blockade of twitch contractions after 10 min was 70% for both toxins. In 1.5
mM Ca2+ omega-conotoxin MVIIC (1 microM) inhibited completely the twitch
contraction after 10 min. In 5 mM Ca2+ blockade developed very slowly and was
very poor after 30 min, omega-conotoxin MVIIC depressed the response by only
20%. These results are compatible with the idea that the three omega-conotoxins
block the purinergic neurotransmission of the vas deferens by acting on
presynaptic N-type voltage-dependent Ca2+ channels. However, omega-conotoxin
MVIIC seems to bind to sites different from those recognised by omega-conotoxin
GVIA and MVIIA, which are markedly differentiated by their Ca2+ requirements for
binding to their receptors. Despite their high sequence homology, the peptide neurotoxins omega-conotoxin
MVIIA and MVIIC selectively block N- and P/Q-type calcium channels,
respectively. To study the recognition mechanism of calcium channel subtypes,
two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by
exchanging their N- and C-terminal halves. Binding assay for both N- and
P/Q-type calcium channels showed that amino acid residues restricted to the
N-terminal half are important for the recognition of N-type channels, whereas
essential residues for P/Q-type channel recognition are widely spread over the
whole omega-conotoxin molecule. Omega-conotoxin MVIIC (MVIIC) blocks P/Q-type calcium channels with high
affinity and N-type calcium channels with low affinity, while the highly
homologous omega-conotoxin MVIIA blocks only N-type calcium channels. We wished
to obtain MVIIC analogues more selective for P/Q-type calcium channels than
MVIIC to elucidate structural differences among the channels, which discriminate
the omega-conotoxins. To prepare a number of MVIIC analogues efficiently, we
developed a combinatorial method which includes a random air oxidation step.
Forty-seven analogues were prepared in six runs and some of them exhibited
higher selectivity for P/Q-type calcium channels than MVIIC in binding assays. Replacement of the N-terminal half of omega-conotoxin MVIIC, a peptide blocker
of P/Q-type calcium channels, with that of omega-conotoxin MVIIA significantly
increased the affinity for N-type calcium channels. To identify the residues
essential for subtype selectivity, we examined single reverse mutations from
MVIIA-type to MVIIC-type in this chimeric analog. A reverse mutation from Lys(7)
to Pro(7) decreased the affinity for both P/Q- and N-type channels, whereas that
from Leu(11) to Thr(11) increased the affinity for P/Q-type channels and
decreased the affinity for N-type channels. The roles of these two residues were
confirmed by synthesizing two MVIIC analogs in which Pro(7) and Thr(11) were
replaced with Lys(7) and Leu(11), respectively. Rises in intracellular Ca2+ induced by activation of glutamate receptors are of
ultimate importance for neuronal excitability and pathophysiological processes.
In the present study, we aimed to elucidate the types of voltage-dependent Ca2+
channels involved in the NMDA-stimulated influx of Ca2+ into the isolated rat
retina by using selective blockers. Additionally, the number of binding sites
for radioligands labelling L- ([3H]nitrendipine), N- ([125I]omega-conotoxin
MVIIA) and P/Q-type ([125I]omega-conotoxin MVIIC) Ca2+ channels was assessed in
the rat retina and, for further comparison, in the rat cortex. Incubation of
isolated rat retinas with 100 microM NMDA produced a three-fold increase in the
influx of 45Ca2+ that was completely blunted by MK-801, a NMDA receptor
antagonist, and partially attenuated (approximately 20%) by tetrodotoxin, a Na+
channel blocker. The L-type Ca2+ channel blocker nifedipine reduced
NMDA-stimulated Ca2+ influx in a dose-related fashion, with a maximum reduction
of approximately 50%. Similar effects were observed with verapamil and
diltiazem. Blockers of N- and P/Q-type Ca2+ channels had no significant effect
on the influx of Ca2+ evoked by NMDA. Co2+, a non-specific Ca2+ channel blocker,
caused an inhibition of NMDA-stimulated Ca2+ influx similar to that of
nifedipine. Therefore, of all voltage-dependent Ca2+ channels, L-type channels
appear to make the greatest contribution (up to 50%) to the NMDA-stimulated
influx of Ca2+ into the isolated rat retina. This finding contrasts with
evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type
channels in NMDA-evoked Ca2+ signals. A comparison of the number of radioligand
binding sites associated with L-, N- or P/Q-type Ca2+ channels in the rat cortex
and retina revealed that such a difference cannot be ascribed to a distinct
expression pattern of these channels in both tissues, although some variations
were found. Interestingly, a different affinity of [3H]nitrendipine for L-type
Ca2+ channels in the rat retina and cortex was observed which may reflect the
expression of different classes of L-type channels in these tissues. The ability
of L-type Ca2+ channel blockers to attenuate NMDA-stimulated Ca2+ influx may
underlie their neuroprotective effects in the retina. Although the etiology of neurodevelopmental mental disorders remains obscure,
converging lines of evidence using animal modeling suggest a critical role for
activity-dependent neurodevelopmental processes during neonatal life. Here, we
report the behavioral effects of a novel technique designed to induce targeted,
transient disruption of activity-dependent processes in early development via
reduction of calcium-mediated neurotransmitter release. We examined the
post-pubertal behavioral effects of neonatal (postnatal day 7) medial prefrontal
cortex infusion of either vehicle or N-type and P/Q-type presynaptic
voltage-dependent calcium channel blockers (omega-conotoxins MVIIA and MVIIC
respectively; 6.8 and 45 pmol infused respectively) in rat pups. In a test of
amphetamine-induced behavioral sensitization, neonatal omega-conotoxin MVIIA
treatment significantly increased locomotion following repeated amphetamine
injections (1.5 mg/kg i.p.) and significantly decreased locomotion following
repeated saline injections relative to animals treated neonatally with vehicle.
However, there was no effect of conotoxin treatment on the long-term expression
of amphetamine sensitization. Neonatal treatment with omega-conotoxins had no
effect on the other behaviors assayed, namely, acoustic startle response,
prepulse inhibition of startle, novelty- and amphetamine-induced (1.5 mg/kg
i.p.) locomotion, and anxiety-like behavior in the elevated plus-maze. These
data confirm that transient, region-specific disruption of synaptic transmission
during early development can have long-term effects on behaviors relevant to
neurodevelopmental mental disorders. |
What is the idea behind the fractal globule that has been proposed as a model of chromatin conformation in the nucleus of a cell? | The fractal globule is a compact polymer state that emerges during polymer condensation as a result of topological constraints which prevent one region of the chain from passing across another one. This long-lived intermediate state was introduced in 1988 (Grosberg et al. 1988) and has not been observed in experiments or simulations until recently (Lieberman-Aiden et al. 2009). Recent characterization of human chromatin using a novel chromosome conformational capture technique brought the fractal globule into the spotlight as a structural model of human chromosome on the scale of up to 10 Mb (Lieberman-Aiden et al. 2009). The fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus is distinct from the more commonly used globular equilibrium model and emphasizes topological constraints as a primary factor driving formation of chromosomal territories. | We describe Hi-C, a method that probes the three-dimensional architecture of
whole genomes by coupling proximity-based ligation with massively parallel
sequencing. We constructed spatial proximity maps of the human genome with Hi-C
at a resolution of 1 megabase. These maps confirm the presence of chromosome
territories and the spatial proximity of small, gene-rich chromosomes. We
identified an additional level of genome organization that is characterized by
the spatial segregation of open and closed chromatin to form two genome-wide
compartments. At the megabase scale, the chromatin conformation is consistent
with a fractal globule, a knot-free, polymer conformation that enables maximally
dense packing while preserving the ability to easily fold and unfold any genomic
locus. The fractal globule is distinct from the more commonly used globular
equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic
conformations of whole genomes. The three-dimensional folding of chromosomes compartmentalizes the genome and
and can bring distant functional elements, such as promoters and enhancers, into
close spatial proximity (2-6). Deciphering the relationship between chromosome
organization and genome activity will aid in understanding genomic processes,
like transcription and replication. However, little is known about how
chromosomes fold. Microscopy is unable to distinguish large numbers of loci
simultaneously or at high resolution. To date, the detection of chromosomal
interactions using chromosome conformation capture (3C) and its subsequent
adaptations required the choice of a set of target loci, making genome-wide
studies impossible (7-10). We developed Hi-C, an extension of 3C that is capable
of identifying long range interactions in an unbiased, genome-wide fashion. In
Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to
one another by means of covalent DNA-protein cross-links. When the DNA is
subsequently fragmented with a restriction enzyme, these loci remain linked. A
biotinylated residue is incorporated as the 5' overhangs are filled in. Next,
blunt-end ligation is performed under dilute conditions that favor ligation
events between cross-linked DNA fragments. This results in a genome-wide library
of ligation products, corresponding to pairs of fragments that were originally
in close proximity to each other in the nucleus. Each ligation product is marked
with biotin at the site of the junction. The library is sheared, and the
junctions are pulled-down with streptavidin beads. The purified junctions can
subsequently be analyzed using a high-throughput sequencer, resulting in a
catalog of interacting fragments. Direct analysis of the resulting contact
matrix reveals numerous features of genomic organization, such as the presence
of chromosome territories and the preferential association of small gene-rich
chromosomes. Correlation analysis can be applied to the contact matrix,
demonstrating that the human genome is segregated into two compartments: a less
densely packed compartment containing open, accessible, and active chromatin and
a more dense compartment containing closed, inaccessible, and inactive chromatin
regions. Finally, ensemble analysis of the contact matrix, coupled with
theoretical derivations and computational simulations, revealed that at the
megabase scale Hi-C reveals features consistent with a fractal globule
conformation. The fractal globule is a compact polymer state that emerges during polymer
condensation as a result of topological constraints which prevent one region of
the chain from passing across another one. This long-lived intermediate state
was introduced in 1988 (Grosberg et al. 1988) and has not been observed in
experiments or simulations until recently (Lieberman-Aiden et al. 2009). Recent
characterization of human chromatin using a novel chromosome conformational
capture technique brought the fractal globule into the spotlight as a structural
model of human chromosome on the scale of up to 10 Mb (Lieberman-Aiden et al.
2009). Here, we present the concept of the fractal globule, comparing it to
other states of a polymer and focusing on its properties relevant for the
biophysics of chromatin. We then discuss properties of the fractal globule that
make it an attractive model for chromatin organization inside a cell. Next, we
connect the fractal globule to recent studies that emphasize topological
constraints as a primary factor driving formation of chromosomal territories. We
discuss how theoretical predictions, made on the basis of the fractal globule
model, can be tested experimentally. Finally, we discuss whether fractal globule
architecture can be relevant for chromatin packing in other organisms such as
yeast and bacteria. Repetitive DNA sequences derived from transposable elements (TE) are distributed
in a non-random way, co-clustering with other classes of repeat elements, genes
and other genomic components. In a previous work we reported power-law-like size
distributions (linearity in log-log scale) in the spatial arrangement of Alu and
LINE1 elements in the human genome. Here we investigate the large-scale features
of the spatial arrangement of all principal classes of TEs in 14 genomes from
phylogenetically distant organisms by studying the size distribution of
inter-repeat distances. Power-law-like size distributions are found to be
widespread, extending up to several orders of magnitude. In order to understand
the emergence of this distributional pattern, we introduce an evolutionary
scenario, which includes (i) Insertions of DNA segments (e.g., more recent
repeats) into the considered sequence and (ii) Eliminations of members of the
studied TE family. In the proposed model we also incorporate the potential for
transposition events (characteristic of the DNA transposons' life-cycle) and
segmental duplications. Simulations reproduce the main features of the observed
size distributions. Furthermore, we investigate the effects of various genomic
features on the presence and extent of power-law size distributions including TE
class and age, mode of parental TE transmission, GC content, deletion and
recombination rates in the studied genomic region, etc. Our observations
corroborate the hypothesis that insertions of genomic material and eliminations
of repeats are at the basis of power-laws in inter-repeat distances. The
existence of these power-laws could facilitate the formation of the recently
proposed "fractal globule" for the confined chromatin organization. Chromatin has a complex spatial organization in the cell nucleus that serves
vital functional purposes. A variety of chromatin folding conformations has been
detected by single-cell imaging and chromosome conformation capture-based
approaches. However, a unified quantitative framework describing spatial
chromatin organization is still lacking. Here, we explore the "strings and
binders switch" model to explain the origin and variety of chromatin behaviors
that coexist and dynamically change within living cells. This simple polymer
model recapitulates the scaling properties of chromatin folding reported
experimentally in different cellular systems, the fractal state of chromatin,
the processes of domain formation, and looping out. Additionally, the strings
and binders switch model reproduces the recently proposed "fractal-globule"
model, but only as one of many possible transient conformations. The fractal globule, a self-similar compact polymer conformation where the chain
is spatially segregated on all length scales, has been proposed to result from a
sudden polymer collapse. This state has gained renewed interest as one of the
prime candidates for the non-entangled states of DNA molecules inside cell
nuclei. Here, we present Monte Carlo simulations of collapsing polymers. We find
through studying polymers of lengths between 500 and 8000 that a chain collapses
into a globule, which is neither fractal, nor as entangled as an equilibrium
globule. To demonstrate that the non-fractalness of the conformation is not just
the result of the collapse dynamics, we study in addition the dynamics of
polymers that start from fractal globule configurations. Also in this case the
chain moves quickly to the weakly entangled globule where the polymer is well
mixed. After a much longer time the chain entangles reach its equilibrium
conformation, the molten globule. We find that the fractal globule is a highly
unstable conformation that only exists in the presence of extra constraints such
as cross-links. |
Which anticancer drugs target human topoisomerase II? | Etoposide (VP-16) and Teniposide (VM-26) are effective as an anti-tumour drug by inhibiting eukaryotic DNA topoisomerase II via establishing a covalent complex with DNA. Doxorubicin, Daunorubicin and Aclarubicin are anthracyclins that act as DNA topoisomerase II inhibitors and may be used in combination. Benzoxazoles, benzimidazoles and related fused heterocyclic compounds, which exhibited significant eukaryotic DNA topoisomerase II inhibitory activity. F14512 is a polyamine-containing epipodophyllotoxin derivative that acts as an inhibitor of DNA topoisomerase II. Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II.
Among topoisomerase II inhibitors, the cytostatic potency was by decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP 941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16 | The effect of combinations of the anthracyclines aclarubicin and daunorubicin
was investigated in a clonogenic assay using the human small cell lung cancer
cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich
ascites tumor (EHR2/DNR+). It was found that the cytotoxicity of daunorubicin in
OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations
of aclarubicin. Coordinately, aclarubicin inhibited the formation of
daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein
cross-links in OC-NYH cells when assayed by the alkaline elution technique.
Aclarubicin had no influence on the accumulation of daunorubicin in these cells.
In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by
more than 300% when the cells were simultaneously incubated with the MDR
modulator verapamil, aclarubicin, or the two agents combined. Yet the
cytotoxicity of daunorubicin was potentiated significantly only by verapamil.
The increased cytotoxicity of daunorubicin in the presence of verapamil was
completely antagonized when aclarubicin was used together with the MDR
modulator. Finally, the effect of daunorubicin on the DNA cleavage activity of
purified topoisomerase II in the presence and absence of aclarubicin was
examined. It was found that daunorubicin stimulated DNA cleavage by
topoisomerase II at specific DNA sites. The addition of aclarubicin completely
inhibited the daunorubicin-induced stimulation of DNA cleavage. Taken together,
these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced
cytotoxicity is due mainly to a drug interaction with the nuclear enzyme
topoisomerase II. This antagonism at the nuclear level explains why aclarubicin
is a poor modulator of daunorubicin resistance even though aclarubicin is able
to increase the intracellular accumulation of daunorubicin in a MDR cell line. In an attempt to clarify the role of drug-induced protein-associated DNA breaks
(i.e., DNA topoisomerase II-mediated DNA cleavage) in the cytotoxic activity of
doxorubicin and etoposide, their cellular effects were compared in 2 human
small-cell lung cancer (SCLC) lines, characterized by differential sensitivity
to DNA topoisomerase II inhibitors. These drugs were selected for comparative
studies since they are among the most effective agents in the treatment of SCLC.
H146 and N592 cell lines were obtained from pleural effusion and bone-marrow
aspirate of pretreated patients, respectively. Both cell lines grew as floating
aggregates with similar doubling times (30 and 33 hr for N592 and H146 cells,
respectively). Although, immediately after 1 hr exposure to equitoxic drug
levels, the extent of DNA cleavage produced by doxorubicin was markedly lower
than that produced by etoposide, DNA lesions produced by doxorubicin persisted
and even increased following drug removal. In contrast, an almost complete
disappearance of etoposide-induced DNA breaks was noted 1 hr after drug removal.
Resealing of strand breaks was faster in N592 than in H146 cells. These findings
suggest that reversal of these lesions plays a major role in cell survival
rather than the occurrence of DNA breaks immediately following drug exposure.
This observation is consistent with the view that inhibition of DNA re-ligation
rather than stimulation of DNA cleavage is the critical step for drug action.
The different response of these cell lines to cytotoxic action of the
topoisomerase inhibitors is associated with a differential drug effect on DNA
integrity (detected as DNA double-strand breaks and DNA-protein cross-links).
However DNA lesions were comparable when cells were exposed to equitoxic drug
levels. The observation that etoposide-induced DNA breaks were similar in
isolated nuclei from both cell lines suggests that drug-target interaction is
modulated in a different manner in the intact cell. As indicated by doxorubicin
uptake and retention, cellular drug pharmacokinetics do not account for the
different drug response of the studied SCLC lines, presumably, reflecting a
different extent of DNA break formation and/or a different cytotoxic consequence
of DNA damage. The effect of combinations of the anthracycline aclarubicin and the
topoisomerase II targeting drugs
4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside)
(VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was
investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost
completely antagonized by preincubating cells with nontoxic concentrations of
aclarubicin. The inhibition of cytotoxicity was not seen when the cells were
exposed to aclarubicin after exposure to VP-16. The inhibition was significant
over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above
which the toxicity of aclarubicin became apparent. A similar effect was seen on
the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with
Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase
II and naked DNA, aclarubicin did not stimulate the formation of cleavable
complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1
microM inhibited the baseline formation of cleavable complexes elicited with the
enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the
formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at
aclarubicin concentrations above 1 microM an antagonistic effect was obtained.
In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks
were completely inhibitable by aclarubicin preincubation with no synergic dose
levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated
DNA cleavage. This inhibition could represent the mechanism of action of the
drug and explain the lack of cross-resistance to the classical anthracyclines.
The observed antagonism could have consequences for scheduling of aclarubicin
with topoisomerase II-active anticancer drugs. As an approach to the rational design of combination chemotherapy involving the
anti-cancer DNA topoisomerase II poison etoposide (VP-16), we have studied the
dynamic changes occurring in small-cell lung cancer (SCLC) cell populations
during protracted VP-16 exposure. Cytometric methods were used to analyse
changes in target enzyme availability and cell cycle progression in a SCLC cell
line, mutant for the tumour-suppressor gene p53 and defective in the ability to
arrest at the G1/S phase boundary. At concentrations up to 0.25 microM VP-16,
cells became arrested in G2 by 24 h exposure, whereas at concentrations 0.25-2
microM G2 arrest was preceded by a dose-dependent early S-phase delay, confirmed
by bromodeoxyuridine incorporation. Recovery potential was determined by
stathmokinetic analysis and was studied further in aphidicolin-synchronised
cultures released from G1/S and subsequently exposed to VP-16 in early S-phase.
Cells not experiencing a VP-16-induced S-phase delay entered G2 delay dependent
upon the continued presence of VP-16. These cells could progress to mitosis
during a 6-24 h period after drug removal. Cells experiencing an early S-phase
delay remained in long-term G2 arrest with greatly reducing ability to enter
mitosis up to 24 h after removal of VP-16. Irreversible G2 arrest was delimited
by the induction of significant levels of DNA cleavage or fragmentation, not
associated with overt apoptosis, in the majority of cells. Western blotting of
whole-cell preparations showed increases in topoisomerase II levels (up to
4-fold) attributable to cell cycle redistribution, while nuclei from cells
recovering from S-phase delay showed enhanced immunoreactivity with an
anti-topoisomerase II alpha antibody. The results imply that traverse of G1/S
and early S-phase in the presence of a specific topoisomerase II poison gives
rise to progressive low-level trapping of topoisomerase II alpha, enhanced
topoisomerase II alpha availability and the subsequent irreversible arrest in G2
of cells showing limited DNA fragmentation. We suggest that protracted, low-dose
chemotherapeutic regimens incorporating VP-16 are preferentially active towards
cells attempting G1/S transition and have the potential for increasing the
subsequent action of other topoisomerase II-targeted agents through target
enzyme modulation. Combination modalities which prevent such dynamic changes
occurring would act to reduce the effectiveness of the VP-16 component. BACKGROUND: The cumulative cardiotoxicity of anthracyclines is thought to result
from the generation of free radicals. New DNA topoisomerase II inhibitors less
prone to redox reactions, such as mitoxantrone and more recently the
anthrapyrazoles, were developed to circumvent this toxicity.
PURPOSE: Two anthrapyrazoles currently in clinical evaluation, DuP 941
(Losoxantrone) and DuP 937, were compared to other topoisomerase II inhibitors
with respect to their cytotoxic potency and selectivity and with respect to
topoisomerase II inhibition.
METHODS: Cytotoxicity was tested in the 60 cell lines of the National Cancer
Institute preclinical antitumor drug discovery screen (NCI screen). The potency
of anthrapyrazoles to inhibit purified topoisomerase II was determined. The
specificity of drug-induced topoisomerase II pattern of cleavage, one of the
cellular determits of cytotoxicity, was investigated in human c-myc DNA.
RESULTS: Using the COMPARE analysis, we found that the most closely related
cytotoxic profiles in the NCI screen were between the anthrapyrazoles and
mitoxantrone. Among topoisomerase II inhibitors, the cytostatic potency was by
decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP
941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16. The
potency of mitoxantrone and anthrapyrazoles to generate DNA double-strand
breaks, by induction of the topoisomerase II cleavable complexes in nuclear
extracts, was in agreement with cytotoxicity. Sequencing of drug-induced
topoisomerase II cleavages in c-myc DNA showed a common cleavage pattern for
anthrapyrazoles and mitoxantrone. This pattern was different from the patterns
obtained with other topoisomerase II inhibitors.
CONCLUSION: At the molecular and cellular levels, anthrapyrazoles are potent
topoisomerase II inhibitors closely related to mitoxantrone.
IMPLICATIONS: These results validate the COMPARE analysis using the NCI screen
to predict molecular mechanisms of drug action. Anthrapyrazoles, which are
unlikely to produce free radicals, might be useful in the same indications as
mitoxantrone, especially for patients with cardiac risks, for pediatric
patients, and for patients treated with intensified protocols. Suramin is a prototype of a new class of anticancer drugs. We investigated the
action of suramin on the signal transduction pathways to DNA topoisomerase II
(Topo II). Suramin showed a growth-inhibitory effect on a human lung cancer cell
line (PC-9) with an IC50 of about 160 micrograms/ml. Suramin inhibited the
catalytic activity of Topo II with an IC50 of about 100 micrograms/ml without
stabilization of the cleavable complex of DNA and Topo II. Suramin decreased the
phosphorylation of Topo II with an IC50 of 175 micrograms/ml, but did not change
the degree of Topo II expression. These IC50 values for inhibition of catalytic
activity and phosphorylation of Topo II were equivalent to the growth-inhibitory
dose determined by tetrazolium dye assay. Phosphorylation of the tyrosine
residues of Topo II was not changed by suramin. In the presence of okadaic acid,
a potent inhibitor of serine/threonine protein phosphatase, suramin also
decreased the phosphorylation of Topo II, suggesting that the drug did not act
on the serine/threonine protein phosphatases inhibited by okadaic acid. Suramin
also inhibited the protein kinase C (PKC) activity of PC-9 cells. These results
suggest that suramin decreases the phosphorylation of Topo II mediated by PKC.
This effect of suramin might cause the inhibition of Topo II activity resulting
in the growth inhibition of tumor cells. We examined whether heat stress could enhance the sensitivity of human colon
cancer WiDr cells to topoisomerase II-targeting anticancer agents, etoposide
(VP-16) and teniposide (VM-26), and also determined the most effective timing
for the drug administration after exposure to hyperthermia. Both topoisomerase
II contents and topoisomerase II activity were significantly increased in WiDr
cells 3 to 12 h after heat stress at 43 degrees C for 1 h, in comparison with
those immediately after the heat stress. Cytotoxicity by VP-16 was most
significantly enhanced 3 to 12 h after exposure to 43 degrees C for 1 h, but no
synergistic effect was observed when the drug was administered immediately after
the heat stress. A combination of VM-26 with heat stress, but not that of a
topoisomerase I-targeting camptothecin derivative (CPT-11), or vincristine,
showed a synergistic cytotoxic effect on WiDr cells. VP-16 alone induced
cellular accumulation at the G2 + M phase, whereas the combination of VP-16 and
heat stress further increased the cell population at the G2 + M phase, and
decreased S-phase cells. A possible application of the combination of VP-16 and
hyperthermia in clinical use is discussed. PURPOSE: Topoisomerase II alpha content, topoisomerase II catalytic activity and
drug sensitivities to the topoisomerase II inhibitors, doxorubicin and
etoposide, were examined in a panel of 14 unselected human lung cancer cell
lines in order to determine the relationship between topoisomerase II and drug
sensitivities to the topoisomerase II inhibitors.
METHODS: Drug sensitivities were determined using a microculture tetrazolium
assay. The topoisomerase II alpha levels were determined by Western blot
analysis and the topoisomerase II catalytic activity was determined using a
decatenation assay of kinetoplast DNA, using nuclear protein from cells of each
cell line.
RESULTS: Drug sensitivity tests revealed that small-cell lung cancer (SCLC) cell
lines were more sensitive to drugs than non-small-cell lung cancer (NSCLC) cell
lines. The relative topoisomerase II alpha levels and relative topoisomerase II
catalytic activity from SCLC cell lines (mean +/- SD 0.89 +/- 0.54 and 5.3 +/-
3.4, respectively) were slightly higher than those from NSCLC cell lines (0.78
+/- 0.56 and 4.0 +/- 2.8, respectively), but the differences were not
statistically significant, and not sufficient to account for the variation in
drug sensitivities. Moreover, no clear association was observed between the
topoisomerase II alpha levels or the topoisomerase II catalytic activity and
drug sensitivities in the cell lines studied.
CONCLUSIONS: These findings suggest that the difference in drug sensitivities to
doxorubicin and etoposide in human lung cancer cell lines might not be
explainable by the topoisomerase II alpha levels and topoisomerase II catalytic
activity. Moreover, our results suggest that the topoisomerase II alpha levels
and topoisomerase II catalytic activity may play a minor role in the
determination of clinical drug resistance of human lung cancers. Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA
topoisomerase II, with at least two effects on the enzyme: namely, locking it in
a closed-clamp form and inhibiting its ATPase activity. This is in contrast to
topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by
stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is
cleaved and the protein is covalently attached to DNA. Human small cell lung
cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25%
increase in topoisomerase IIalpha level and no change in expression of the beta
isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells
demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a
R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in
the alpha isoform), this being the first drug-selected mutation described at
this site. Western blotting after incubation with ICRF-187 showed no depletion
of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells,
whereas equal depletion of the beta isoform was observed in the two sublines.
Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced
DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily
apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha
introduced into a yeast Saccharomyces cerevisiae strain with a
temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred
resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or
m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q
enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic
capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to
the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not
resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than
wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in
the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by
a decreased ATP binding by the mutated enzyme that is responsible for
bisdioxopiperazine resistance. Gastric cancer is poorly-responsive to widely used antitumour drugs, the
efficacy of which is thought to be related to the capacity of triggering
apoptosis. This process requires a series of gene products including a
functional p53 protein. We tested the effects of two DNA topoisomerase II
poisons, etoposide and doxorubicin, on gastric cancer cell lines with different
genetic lesions. We characterised MKN74 and MKN28 cells for p53 gene status and
for the expression of p53 and p21 proteins, as well as of topoisomerase II alpha
and beta isoforms. After drug treatments, the cells were analysed for drug
cytotoxicity, colony forming ability, cell cycle distribution and presence of
apoptotic features. Our findings demonstrated that both etoposide and
doxorubicin have a potent anti-proliferative effect on gastric cancer cells.
Cell death kinetics was different in the two cell lines, MKN74 cells being more
sensitive than MKN28 to the drugs. MKN74 cells, although harboring a wt p53
gene, were unable to undergo a massive apoptosis following etoposide treatment.
The response of this cell line might be related to the topoisomerase II beta
isozyme, the expression of which proved to be undetectable. We have prepared full-length Drosophila and human topoisomerase II and
truncation constructs containing the amino-terminal ATPase domain, and we have
analyzed their biochemical properties. The ATPase activity of the truncation
proteins, similar to that of the full-length proteins, is greatly stimulated by
the presence of DNA. This activity of the truncation proteins is also sensitive
to the inhibition by the drug bisdioxopiperazine, ICRF-193, albeit at a much
lower level than the full-length protein. Therefore, bisdioxopiperazine can
directly interact with the NH(2)-terminal ATPase domain, but the drug-enzyme
interaction may involve other domains as well. The ATPase activity of the ATPase
domain protein showed a quadratic dependence on enzyme concentration, suggesting
that dimerization of the NH(2)-terminal domain is a rate-limiting step. Using
both protein cross-linking and sedimentation equilibrium analysis, we showed
that the ATPase domain exists as a monomer in the absence of cofactors but can
readily dimerize in the presence of a nonhydrolyzable analog of ATP,
5'-adenylyl-beta,gamma-imidodiphosphate. More interestingly, both ATP and ADP
can also promote protein dimerization. This result thus suggests that the
protein clamp, mediated through the dimerization of ATPase domain, remains
closed after ATP hydrolysis and opens upon the dissociation of ADP. Twenty previously synthesized fused heterocyclic DNA-topoisomerase II (Topo
II)-inhibiting compounds were investigated for their potential efficacy in
various human cancer cell lines that were derived from different tumor entities.
Moreover, different multidrug-resistant variants of these cancer cell lines with
decreased Topo II expression were investigated. In parental, drug-sensitive
cells merely the compounds BD3 and G35 showed efficacies, in terms of microM,
which were similar to that of the classical Topo II inhibitor etoposide. On the
other hand, most of the tested heterocyclic compounds were found more effective
in drug-resistant cells than in the parental, drug-sensitive ones, and some of
the compounds showed high antineoplastic efficacy in several drug-resistant cell
models. Compounds BD13, BD14 and BD16 exhibited high antineoplastic activities
against the drug-resistant sublines EPG85-257RNOV and EPG85-257RDB derived from
gastric carcinoma, EPP85-181RNOV and EPP85-181RDB derived from pancreatic
carcinoma, MCF-7/Adr derived from breast cancer, D79/86RNOV derived from
fibrosarcoma, and MeWoETO1 derived from melanoma. Furthermore, compound D23 was
found highly efficient in the multidrug-resistant variants HT-29RNOV and
HT-29RDB derived from colon carcinoma, and compound D24 exhibited the highest
antineoplastic activity among the tested compounds in the drug-resistant subline
MDA-MB-231ROV derived from breast cancer. In conclusion, compounds BD 13, BD 14,
BD 16, D 23 and D 24 may be useful for the treatment of different
multidrug-resistant cancer cells with cross resistance against "classical" Topo
II-targeting drugs. The polyamine transport system (PTS) is an energy-dependent machinery frequently
overactivated in cancer cells with a high demand for polyamines. We have
exploited the PTS to selectively deliver a polyamine-containing drug to cancer
cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II
with a spermine moiety introduced as a cell delivery vector. The polyamine tail
supports three complementary functions: (a) facilitate formulation of a
water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II
inhibition, and (c) facilitate selective uptake by tumor cells via the PTS.
F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with
CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210
leukemia cells to F14512 was observed in the presence of putrescine, spermidine,
and spermine. In parallel, the spermine moiety considerably enhances the
drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II.
The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA
anchor, and this property translates at the cellular level into a distinct
pharmacologic profile. Twenty-nine human solid or hematologic cell lines were
used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18
micromol/L). Finally, the potent antitumor activity of F14512 in vivo was
evidenced with a MX1 human breast tumor xenograft model, with partial and
complete tumor regressions. This work supports the clinical development of
F14512 as a novel targeted cytotoxic drug and sheds light on the concept of
selective delivery of drugs to tumor cells expressing the PTS. PURPOSE: Human epidermal growth factor receptor 2 (HER2)/neu, topoisomerase II
alpha (TOP2A), and polysomy 17 may predict tumor responsiveness to doxorubicin
(DOX) therapy.
METHODS: We identified neoadjuvant DOX/cyclophosphamide treated breast cancer
patients in our registry from 1997 to 2008 with sufficient tissue for testing (n
= 34). Fluorescence in situ hybridization (FISH) testing was done on
deparaffinized tissue sections pretreated using vendor's standard protocol
modification, and incubated with US Food and Drug Administration approved Abbott
Diagnostics Vysis PathVysion™ probe set, including Spectrum-Green-conjugated
probe to α-satellite DNA located at the centromere of chromosome 17
(17p11.1-q11.1) and a Spectrum-Orange-conjugated probe to the TOP2A gene.
Morphometric analysis was performed using a MetaSystems image analysis system.
Manual counting was performed on all samples in which autofluorescence and/or
artifact prevented the counting of sufficient numbers of cells. A ratio >2.0 was
considered positive for TOP2A amplification. Polysomy 17 (PS17) presence was
defined as signals of ≥2.5. Outcomes were pathological complete response (pCR),
partial response (PR), and nonresponse (NR).
RESULTS: Of 34 patients tested, one was TOP2A amplified (hormone receptor
negative/HER2 negative, partial responder). The subset of TOP2A nonamplified,
HER2 negative, and PS17 absent (n = 23) patients had treatment response: pCR = 2
(9%), PR = 14 (61%), and NR = 7 (30%). Including the two PS17 present and
HER2-positive patients (n = 33), 76% of TOP2A nonamplified patients had pCR or
PR.
CONCLUSIONS: We observed substantial treatment response in patients lacking
three postulated predictors that would be difficult to attribute to
cyclophosphamide alone. Patients who are HER2 negative and lack TOP2A
amplification and PS17 should not be excluded from receiving DOX-containing
regimens. Etoposide is effective as an anti-tumour drug by inhibiting eukaryotic DNA
topoisomerase II via establishing a covalent complex with DNA. Unfortunately,
its wide therapeutic application is often hindered by multidrug resistance
(MDR), low water solubility and toxicity. In our previous study, new derivatives
of benzoxazoles, benzimidazoles and related fused heterocyclic compounds, which
exhibited significant eukaryotic DNA topoisomerase II inhibitory activity, were
synthesized and exhibited better inhibitory activity compared with the drug
etoposide itself. To expose the binding interactions between the eukaryotic
topoisomerase II and the active heterocyclic compounds, docking studies were
performed, using the software Discovery Studio 2.1, based on the crystal
structure of the Topo IIA-bound G-segment DNA (PDB ID: 2RGR). The research was
conducted on a selected set of 31 fused heterocyclic compounds with variation in
structure and activity. The structural analyses indicate coordinate and hydrogen
bonding interactions, van der Waals interactions and hydrophobic interactions
between ligands and the protein, as Topo IIA-bound G-segment DNA are responsible
for the preference of inhibition and potency. Collectively, the results
demonstrate that the compounds 1a, 1c, 3b, 3c, 3e and 4a are significant
anti-tumour drug candidates that should be further studied. |
What is SHAPE-Seq? | SHAPE-Seq is a high-throughput technique that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. SHAPE-Seq has the ability to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems. SHAPE-Seq v2.0 is a 'universal' method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. It is a highly reproducible method, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. SHAPE-Seq v2.0 is expected to be broadly applicable to understanding the RNA sequence-structure relationship at the heart of some of life's most fundamental processes. | New regulatory roles continue to emerge for both natural and engineered
noncoding RNAs, many of which have specific secondary and tertiary structures
essential to their function. Thus there is a growing need to develop
technologies that enable rapid characterization of structural features within
complex RNA populations. We have developed a high-throughput technique,
SHAPE-Seq, that can simultaneously measure quantitative, single
nucleotide-resolution secondary and tertiary structural information for hundreds
of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl
acylation analyzed by primer extension (SHAPE) chemistry with multiplexed
paired-end deep sequencing of primer extension products. This generates millions
of sequencing reads, which are then analyzed using a fully automated data
analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq
experiment. We demonstrate the ability of SHAPE-Seq to accurately infer
secondary and tertiary structural information, detect subtle conformational
changes due to single nucleotide point mutations, and simultaneously measure the
structures of a complex pool of different RNA molecules. SHAPE-Seq thus
represents a powerful step toward making the study of RNA secondary and tertiary
structures high throughput and accessible to a wide array of scientific
pursuits, from fundamental biological investigations to engineering RNA for
synthetic biological systems. Sequence census methods reduce molecular measurements such as transcript
abundance and protein-nucleic acid interactions to counting problems via DNA
sequencing. We focus on a novel assay utilizing this approach, called selective
2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), that
can be used to characterize RNA secondary and tertiary structure. We describe a
fully automated data analysis pipeline for SHAPE-Seq analysis that includes read
processing, mapping, and structural inference based on a model of the
experiment. Our methods rely on the solution of a series of convex optimization
problems for which we develop efficient and effective numerical algorithms. Our
results can be easily extended to other chemical probes of RNA structure, and
also generalized to modeling polymerase drop-off in other sequence census-based
experiments. Knowledge of RNA structure is critical to understanding both the important
functional roles of RNA in biology and the engineering of RNA to control
biological systems. This article contains a protocol for selective 2'-hydroxyl
acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through
a combination of structure-dependent chemical probing and next-generation
sequencing technologies, achieves structural characterization of hundreds of
RNAs in a single experiment. This protocol is applicable in a variety of
conditions, and represents an important tool for understanding RNA biology. The
protocol includes methods for the design and synthesis of RNA mixtures for
study, and the construction and analysis of structure-dependent sequencing
libraries that reveal structural information of the RNAs in the mixtures. The
methods are generally applicable to studying RNA structure and interactions in
vitro in a variety of conditions, and allows for the rapid characterization of
RNA structures in a high-throughput manner. Curr. Protoc. Chem. Biol. 4:275-297
© 2012 by John Wiley & Sons, Inc. RNA structure is a primary determit of its function, and methods that merge
chemical probing with next generation sequencing have created breakthroughs in
the throughput and scale of RNA structure characterization. However, little work
has been done to examine the effects of library preparation and sequencing on
the measured chemical probe reactivities that encode RNA structural information.
Here, we present the first analysis and optimization of these effects for
selective 2'-hydroxyl acylation analyzed by primer extension sequencing
(SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly
reproducible reactivity data over a wide range of RNA structural contexts with
no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a
'universal' method that can obtain reactivity information for every nucleotide
of an RNA without having to use or introduce a specific reverse transcriptase
priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible,
with reactivity data that can be used as constraints in RNA folding algorithms
to predict structures on par with those generated using data from other SHAPE
methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding
the RNA sequence-structure relationship at the heart of some of life's most
fundamental processes. |
Which are the known inhibitors of the TPL2/MAP3K8 protein? | [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles were the first Tumor Progression Loci-2 (Tpl2) kinase inhibitors. 4-alkylamino-[1,7]naphthyridine-3-carbonitriles are also known to inhibit Tpl2 function as well as quinoline-3-carbonitrile derivatives, thieno[3,2-d]pyrimidines and 2,4-disubstituted thieno[2,3-c]pyridines, indazoles, 4-Alkylamino-[1,7]naphthyridine-3-carbonitriles and generally molecules belonging to the wide categories of quinoline-3-carbonitriles, indazoles and thieno-pyrimidines. | The synthesis and structure-activity studies of a series of
6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of
Tpl2 kinase are described. The early exploratory work described here may lead to
the discovery of compounds with significant therapeutic potential for treating
rheumatoid arthritis and other inflammatory diseases. Tumor progression loci-2 (Tpl2) (Cot/MAP3K8) is a serine/threonine kinase in the
MAP3K family directly upstream of MEK. Recent studies using Tpl2 knockout mice
have indicated an important role for Tpl2 in the lipopolysaccharide (LPS)
induced production of tumor necrosis factor alpha (TNF-alpha) and other
proinflammatory cytokines involved in diseases such as rheumatoid arthritis.
Initial 4-anilino-6-aminoquinoline-3-carbonitrile leads showed poor selectivity
for Tpl2 over epidermal growth factor receptor (EGFR) kinase. Using molecular
modeling and crystallographic data of the EGFR kinase domain with and without an
EGFR kinase-specific 4-anilinoquinazoline inhibitor (erlotinib, Tarceva), we
hypothesized that we could diminish the inhibition of EGFR kinase by
substitution at the C-8 position of our
4-anilino-6-aminoquinoline-3-carbonitrile leads. The
8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles were prepared from the
appropriate 2-substituted 4-nitroanilines. Modifications to the C-6 and C-8
positions led to the identification of compounds with increased inhibition of
TNF-alpha release from LPS-stimulated rat and human blood, and these analogues
were also highly selective for Tpl2 kinase over EGFR kinase. Further
structure-activity based modifications led to the identification of
8-bromo-4-(3-chloro-4-fluorophenylamino)-6-[(1-methyl-1H-imidazol-4-yl)methylamino]quinoline-3-carbonitrile,
which demonstrated in vitro as well as in vivo efficacy in inhibition of
LPS-induced TNF-alpha production. Evaluation of hit chemotypes from high throughput screening identified a novel
series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors.
Structural modifications exploring SAR at the 2- and 4-positions resulting in
inhibitors with improved enzyme potency and cellular activity are disclosed. COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production
of TNF-alpha and other pro-inflammatory cytokines such as IL-1beta via the
ERK/MAP kinase pathway. As TNF-alpha and IL-1beta are clinically validated
targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT
provides a potential avenue for amelioration of disease. Herein we describe
identification of a cellular active selective small molecule inhibitor of COT
kinase. Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an
important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and
signaling. We have discovered that
8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles
(4) are potent inhibitors of this enzyme. In order to improve the inhibition of
TNF-alpha production in LPS-stimulated human blood, a series of analogs with a
variety of substitutions around the triazole moiety were studied. We found that
a cyclic amine group appended to the triazole ring could considerably enhance
potency, aqueous solubility, and cell membrane permeability. Optimization of
these cyclic amine groups led to the identification of
8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile
(34). In a LPS-stimulated rat inflammation model, compound 34 showed good
efficacy in inhibiting TNF-alpha production. The activation of mitogen-activated protein kinases (MAPKs) is critically
involved in inflammatory events through mediation of the production of various
inflammatory cytokines. The Tpl2 (tumor progression locus 2)-MEK (MAPK/ERK
kinase)-ERK (extracellular signal-regulated kinase) signaling pathway plays an
essential role in the production of tumor necrosis factor alpha (TNFalpha) in
macrophages stimulated with lipopolysaccharide (LPS). Here, we studied the
molecular mechanisms of Tpl2-mediated TNFalpha production using a potent Tpl2
kinase inhibitor, 1,7-naphtyridine-3-carbonitrile, and LPS-stimulated RAW264.7
cells. This inhibitor was effective in suppressing the in vitro Tpl2 kinase
activity, and caused a significant reduction in TNFalpha production via specific
suppression of the phosphorylation of MEK and ERK but not that of p38 and c-Jun
N-terminal kinase (JNK). A p38 inhibitor, SB203580, also inhibited the TNFalpha
production dose-dependently. Although the TNFalpha mRNA level was not altered by
either inhibitor, the Tpl2 inhibitor increased the nuclear TNFalpha mRNA level,
while decreasing that in the cytoplasm. Tip-associated protein (TAP), a key
molecule in the nucleocytoplasmic transport of TNFalpha mRNA, was up-regulated
by LPS, but this increase was impaired by the Tpl2 inhibitor. In all cases,
SB203580 was without effect in the presence of LPS. These results suggest that
the LPS-induced TNFalpha production via the Tpl2-MEK-ERK signaling pathway is
regulated by changing the TAP level at the nucleocytoplasmic transport level.
These results improve understanding of TNFalpha regulatory mechanisms and might
provide a new therapeutic strategy against inflammatory diseases. Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a
promising strategy for developing chemopreventive agents, because TNF-α-mediated
cyclooxygenase (COX)-2 expression plays a key role in inflammation and
carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone]
exerts anticarcinogenic effects, although little is known about the underlying
molecular mechanisms and specific targets of this compound. In the present
study, we found that luteolin inhibited TNF-α-induced COX-2 expression by
down-regulating the transactivation of nuclear factor-κB and activator
protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of
mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)
kinase 1/ERK/p90(RSK), mitogen-activated protein kinase kinase 4/c-Jun
N-terminal kinase/c-Jun, and Akt/p70(S6K). However, it had no effect on the
phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling
pathways and COX-2 expression are similar to those achieved by blocking tumor
progression locus 2 serine/threonine kinase (TPL2) using pharmacologic
inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro
and in TPL2 immunoprecipitation kinase assays by binding directly in an
ATP-competitive manner. Overall, these results indicate that luteolin exerts
potent chemopreventive activities, which primarily target TPL2. |
What is the mode of inheritance in Fanconi anemia? | Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished so far. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked | The formal genetics of Fanconi's anemia were investigated on the basis of 21
families from different European countries, and of 69 families from the
literature.
CONCLUSIONS: 1. The result of segregation analysis is compatible with the
hypothesis of a simple autosomal recessive mode of inheritance. 2. The number of
sporadic cases is not greater than expected. 3. Among the affected siblings in
the sibships analyzed, males are somewhat more frequent than females. However,
this sex difference is also found among the unaffected siblings, and it is not
statistically significant. 4. Contrary to assertions made in the literature,
there is no clustering of affected in the sequence of siblings, no maternal age
effect, and no preference of higher birth orders. 5. A high intrafamilial
correlation for age at onset, and (very probably) number and severity of
malformations points to genetic heterogeneity. Apart from the standard type, an
especially mild type with late onset, few malformations, and a relatively benign
course seems to exist. Its counterpart is possibly an especially severe type
with early onset, many malformations, and a maligt course. However, definite
conclusions on the special character of this heterogeneity will require
application of additional methods. The familial occurrence of duodenal atresia is uncommon. This study evaluated
the inheritance patterns, the nature and associations, and the presence of
immunologic deficits in duodenal atresia recurring in at least three siblings
each in two nonrelated families. In the first family, an association with
Fanconi's anemia was observed in three of seven pregcies (2 boys, 1 girl)
suggesting an autosomal recessive mode of transmission. Patients died as a
result of overwhelming (fungal) septicemia in association with pancytopenia. In
a second family, identical multiple atresias occurred in two female siblings
born 18 months apart and a third child with a duodenal stenosis. Overwhelming
sepsis and a T-cell dysfunction was seen in the postoperative period, which had
partially corrected by follow-up at 5 months. A history of family occurrence of
duodenal atresia should alert the physician to the possibility of associated
pathology including immune deficiency states. Fanconi anemia is an autosomal recessive syndrome characterized by diverse
clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal
instability and susceptibility to cancer. Fanconi anemia has at least 11
complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in
8 of these have been identified. The gene BRCA2 was suggested to underlie
complementation group B, but the evidence is inconclusive. Here we show that the
protein defective in individuals with Fanconi anemia belonging to
complementation group B is an essential component of the nuclear protein 'core
complex' responsible for monoubiquitination of FANCD2, a key event in the
DNA-damage response pathway associated with Fanconi anemia and BRCA.
Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and
subject to X-chromosome inactivation. X-linked inheritance has important
consequences for genetic counseling of families with Fanconi anemia belonging to
complementation group B. Its presence as a single active copy and essentiality
for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable
component of the cellular machinery that maintains genomic integrity. Fanconi anemia (FA), a recessive syndrome with both autosomal and X-linked
inheritance, features diverse clinical symptoms, such as progressive bone marrow
failure, hypersensitivity to DNA cross-linking agents, chromosomal instability
and susceptibility to cancer. At least 12 genetic subtypes have been described
(FA-A, B, C, D1, D2, E, F, G, I, J, L, M) and all except FA-I have been linked
to a distinct gene. Most FA proteins form a complex that activates the FANCD2
protein via monoubiquitination, while FANCJ and FANCD1/BRCA2 function downstream
of this step. The FA proteins typically lack functional domains, except for
FANCJ/BRIP1 and FANCM, which are DNA helicases, and FANCL, which is probably an
E3 ubiquitin conjugating enzyme. Based on the hypersensitivity to cross-linking
agents, the FA proteins are thought to function in the repair of DNA interstrand
cross-links, which block the progression of DNA replication forks. Here we
present a hypothetical model, which not only describes the assembly of the FA
pathway, but also positions this pathway in the broader context of DNA
cross-link repair. Finally, the possible role for the FA pathway, in particular
FANCF and FANCB, in the origin of sporadic cancer is discussed. Fanconi anemia (FA) is a rare genetic disease with both autosomal and X-linked
inheritance, characterized by genomic instability. The cells from individuals
with FA are highly sensitive to DNA-crosslinking drugs, such as mitomycin C
(MMC), diepoxybutane (DEB) and so on. Now at least 13 genes (FA-A, B, C, D1, D2,
E, F, G, I, J, L, M, N) have been identified, whose products participate in a
DNA damage-response network involving breast cancer susceptibility gene
products, BRCA1 and BRCA2. The impaired DNA repair due to mutations in FA genes
is thought to be one of the main pathogenesis of FA, also closely related to the
development of some cancers. In this review, the advances of study about FA-BRCA
network are summarized. Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms
including developmental defects, short stature, bone marrow failure, and a high
risk of maligcies. Fifteen genetic subtypes have been distinguished so far.
The mode of inheritance for all subtypes is autosomal recessive, except for
FA-B, which is X-linked. Cells derived from FA patients are-by
definition-hypersensitive to DNA cross-linking agents, such as mitomycin C,
diepoxybutane, or cisplatinum, which becomes manifest as excessive growth
inhibition, cell cycle arrest, and chromosomal breakage upon cellular exposure
to these drugs. Here we provide a detailed laboratory protocol for the accurate
assessment of the FA diagnosis as based on mitomycin C-induced chromosomal
breakage analysis in whole-blood cultures. The method also enables a
quantitative estimate of the degree of mosaicism in the lymphocyte compartment
of the patient. Fanconi anemia (FA) is a rare genetic disease, transmitted in an autosomal
recessive mode. The clinical phenotype is very broad and heterogeneous, related
to the wide range of genes involved in this pathology. The classical triad of
short height, physical abnormalities, and bone marrow failure is suggestive. The
main physical abnormalities found involve the limbs, spinal column, skin,
kidneys and urinary tract, and the ORL zone. Recent progress in molecular
biology has identified 15 genes whose mutation causes FA chromosomal
instability. FA is diagnosed by cytogenetic examination, then specified by
molecular analysis. As FA patients may present multiorgan abnormalities and a
high risk for neoplasia development, their medical follow-up has to be
multidisciplinary and prolonged throughout life. The main challenges of the
follow-up are patient information and education. Bone marrow failure, appearing
during the first decade, requires close hematological monitoring and for severe
cases requires hematopoietic stem cell transplantation, major and specific care
with frequent serious complications and high mortality, but this is the only
curative treatment in FA. Extrahematological care consists in screening for
organ abnormalities and defects as well as monitoring precancerous lesions and
tumors. |
Describe the involvement of conserved noncoding sequences in the regulation of Hox genes. | Comparisons of noncoding sequences of the elephant shark and human Hox clusters have identified a large number of conserved noncoding elements (CNEs), which represent putative cis-regulatory elements that may be involved in the regulation of Hox genes. The b-paralogs of the duplicated fugu Hox clusters are virtually devoid of unique ancient CNEs. Elephant shark and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are indeed enhancers directing tissue-specific expression of Hox genes, divergence of their sequences in vertebrate lineages might have led to altered expression patterns and presumably the functions of their associated Hox genes. When compared, the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters were found to have several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus. | Reconstructing the evolutionary history of Hox cluster origins will lead to
insights into the developmental and evolutionary significance of Hox gene
clusters in vertebrate phylogeny and to their role in the origins of various
vertebrate body plans. We have isolated two Hox clusters from the horn shark,
Heterodontus francisci. These have been sequenced and compared with one another
and with other chordate Hox clusters. The results show that one of the horn
shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a
structural similarity to the amphioxus cluster, whereas the other shark cluster
(HoxN) is orthologous to the mammalian HoxD cluster based on cluster
organization and a comparison with noncoding and Hox gene-coding sequences. The
persistence of an identifiable HoxA cluster over an 800-million-year divergence
time demonstrates that the Hox gene clusters are highly integrated and
structured genetic entities. The data presented herein identify many noncoding
sequence motifs conserved over 800 million years that may function as genetic
control motifs essential to the developmental process. Comparisons of DNA sequences among evolutionarily distantly related genomes
permit identification of conserved functional regions in noncoding DNA. Hox
genes are highly conserved in vertebrates, occur in clusters, and are
uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of
the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark,
human, and mouse, which are separated by approximately 500 million years of
evolution. In support of our approach, several identified putative regulatory
elements known to regulate the expression of Hox genes were recovered. The
majority of the newly identified putative regulatory elements contain short
fragments that are almost completely conserved and are identical to known
binding sites for regulatory proteins (Transfac database). The regulatory
intergenic regions located between the genes that are expressed most anteriorly
in the embryo are longer and apparently more evolutionarily conserved than those
at the other end of Hox clusters. Different presumed regulatory sequences are
retained in either the Aalpha or Abeta duplicated Hox clusters in the fish
lineages. This suggests that the conserved elements are involved in different
gene regulatory networks and supports the duplication-deletion-complementation
model of functional divergence of duplicated genes. The study of Hox clusters and genes provides insights into the evolution of
genomic regulation of development. Derived ray-finned fishes (Actinopterygii,
Teleostei) such as zebrafish and pufferfish possess duplicated Hox clusters that
have undergone considerable sequence evolution. Whether these changes are
associated with the duplication(s) that produced extra Hox clusters is
unresolved because comparison with basal lineages is unavailable. We sequenced
and analyzed the HoxA cluster of the bichir (Polypterus senegalus), a
phylogenetically basal actinopterygian. Independent lines of evidence indicate
that bichir has one HoxA cluster that is mosaic in its patterns of noncoding
sequence conservation and gene retention relative to the HoxA clusters of human
and shark, and the HoxAalpha and HoxAbeta clusters of zebrafish, pufferfish, and
striped bass. HoxA cluster noncoding sequences conserved between bichir and
euteleosts indicate that novel cis-sequences were acquired in the stem
actinopterygians and maintained after cluster duplication. Hence, in the
earliest actinopterygians, evolution of the single HoxA cluster was already more
dynamic than in human and shark. This tendency peaked among teleosts after HoxA
cluster duplication. The posterior HoxA and HoxD genes are essential in appendicular development.
Studies have demonstrated that a "distal limb enhancer," remotely located
upstream of the HoxD complex, is required to drive embryonic autopod expression
of the posterior Hox genes as well as the two additional non-Hox genes in the
region: Evx2 and Lnp. Our work demonstrates a similar mode of regulation for
Hoxa13 and four upstream genes: Evx1, Hibadh, Tax1bp, and Jaz1. These genes all
show embryonic (E11.5-E13.5) distal limb and genital bud expression, suggesting
the existence of a nearby enhancer influencing the expression of a domain of
genes. Comparative sequence analysis between homologous human and mouse genomic
sequence upstream of Hoxa13 revealed a remote 2.25-kb conserved noncoding
sequence (mmA13CNS) within the fourth intron of the Hibadh gene. mmA13CNS shares
a common 131-bp core identity within a conserved noncoding sequence upstream of
Hoxd13, which is located within the previously identified distal limb enhancer
critical region. To test the function of this conserved sequence, we created
mmA13CNS-Hsp86-lacZ transgenic mice. mmA13CNS directed a wide range of tissue
expression, including the central nervous system, developing olfactory tissue,
limb, and genital bud. Limb and genital bud expression directed by mmA13CNS is
not identical to the patterns exhibited by Hoxa13/Evx1/Hibadh/Tax1bp1/Jaz1,
suggesting that mmA13CNS is not sufficient to fully recapitulate their
expression in those tissues. The Evx1- and Evx2-like central nervous system
expression observed in these mice suggests that the long-range regulatory
element(s) for the Hox cluster existed before the cluster duplication. Homeotic (Hox) genes are usually clustered and arranged in the same order as
they are expressed along the anteroposterior body axis of metazoans. The
mechanistic explanation for this colinearity has been elusive, and it may well
be that a single and universal cause does not exist. The Hox-gene complex
(HOM-C) has been rearranged differently in several Drosophila species, producing
a striking diversity of Hox gene organizations. We investigated the genomic and
functional consequences of the two HOM-C splits present in Drosophila buzzatii.
Firstly, we sequenced two regions of the D. buzzatii genome, one containing the
genes labial and abdominal A, and another one including proboscipedia, and
compared their organization with that of D. melanogaster and D. pseudoobscura in
order to map precisely the two splits. Then, a plethora of conserved noncoding
sequences, which are putative enhancers, were identified around the three Hox
genes closer to the splits. The position and order of these enhancers are
conserved, with minor exceptions, between the three Drosophila species. Finally,
we analyzed the expression patterns of the same three genes in embryos and
imaginal discs of four Drosophila species with different Hox-gene organizations.
The results show that their expression patterns are conserved despite the HOM-C
splits. We conclude that, in Drosophila, Hox-gene clustering is not an absolute
requirement for proper function. Rather, the organization of Hox genes is
modular, and their clustering seems the result of phylogenetic inertia more than
functional necessity. The single amphioxus Hox cluster contains 15 genes and may well resemble the
ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of
the European amphioxus Branchiostoma lanceolatum and compared it to the American
species, Branchiostoma floridae, by phylogenetic footprinting to gain insights
into the evolution of Hox gene regulation in chordates. We found that Hox
intergenic regions are largely conserved between the two amphioxus species,
especially in the case of genes located at the 3' of the cluster, a trend
previously observed in vertebrates. We further compared the amphioxus Hox
cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several
conserved noncoding regions, both in intergenic and intronic regions. This
suggests that the regulation of Hox genes is highly conserved across chordates,
consistent with the similar Hox expression patterns in vertebrates and
amphioxus. We have sequenced and analyzed Hox gene clusters from elephant shark, a
holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with
45 Hox genes that include orthologs for a higher number of ancient gnathostome
Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in
teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7
paralogous groups that contain all of the 4 members indicated an ((AB)(CD))
topology for the order of Hox cluster duplication, providing support for the 2R
hypothesis (i.e., 2 rounds of whole-genome duplication during the early
evolution of vertebrates). Comparisons of noncoding sequences of the elephant
shark and human Hox clusters have identified a large number of conserved
noncoding elements (CNEs), which represent putative cis-regulatory elements that
may be involved in the regulation of Hox genes. Interestingly, in fugu more than
50% of these ancient CNEs have diverged beyond recognition in the duplicated
(HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters.
Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually
devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark
and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are
indeed enhancers directing tissue-specific expression of Hox genes, divergence
of their sequences in vertebrate lineages might have led to altered expression
patterns and presumably the functions of their associated Hox genes. It is now well established that there were four Hox gene clusters in the genome
of the last common ancestor of extant gnathostomes. To better understand the
evolution of the organization and expression of these genomic regions, we have
studied the Hox gene clusters of a shark (Scyliorhinus canicula). We sequenced
225,580 expressed sequence tags from several embryonic cDNA libraries. Blast
searches identified corresponding transcripts to almost all the HoxA, HoxB, and
HoxD cluster genes. No HoxC transcript was identified, suggesting that this
cluster is absent or highly degenerate. Using Hox gene sequences as probes, we
selected and sequenced seven clones from a bacterial artificial chromosome
library covering the complete region of the three gene clusters. Mapping of
cDNAs to these genomic sequences showed extensive alternative splicing and
untranslated exon sharing between neighboring Hox genes. Homologous noncoding
exons could not be identified in transcripts from other species using sequence
similarity. However, by comparing conserved noncoding sequences upstream of
these exons in different species, we were able to identify homology between some
exons. Some alternative splicing variants are probably very ancient and were
already coded for by the ancestral Hox gene cluster. We also identified several
transcripts that do not code for Hox proteins, are probably not translated, and
all but one are in the reverse orientation to the Hox genes. This survey of the
transcriptome of the Hox gene clusters of a shark shows that the high complexity
observed in mammals is a gnathostome ancestral feature. Teleost fishes have extra Hox gene clusters owing to shared or lineage-specific
genome duplication events in rayfinned fish (actinopterygian) phylogeny. Hence,
extrapolating between genome function of teleosts and human or even between
different fish species is difficult. We have sequenced and analyzed Hox gene
clusters of the Senegal bichir (Polypterus senegalus), an extant representative
of the most basal actinopterygian lineage. Bichir possesses four Hox gene
clusters (A, B, C, D); phylogenetic analysis supports their orthology to the
four Hox gene clusters of the gnathostome ancestor. We have generated a
comprehensive database of conserved Hox noncoding sequences that include
cartilaginous, lobe-finned, and ray-finned fishes (bichir and teleosts). Our
analysis identified putative and known Hox cis-regulatory sequences with
differing depths of conservation in Gnathostoma. We found that although bichir
possesses four Hox gene clusters, its pattern of conservation of noncoding
sequences is mosaic between outgroups, such as human, coelacanth, and shark,
with four Hox gene clusters and teleosts, such as zebrafish and pufferfish, with
seven or eight Hox gene clusters. Notably, bichir Hox gene clusters have been
invaded by DNA transposons and this trend is further exemplified in teleosts,
suggesting an as yet unrecognized mechanism of genome evolution that may explain
Hox cluster plasticity in actinopterygians. Taken together, our results suggest
that actinopterygian Hox gene clusters experienced a reduction in selective
constraints that surprisingly predates the teleost-specific genome duplication. Cyclostomes, comprising jawless vertebrates such as lampreys and hagfishes, are
the sister group of living jawed vertebrates (gnathostomes) and hence an
important group for understanding the origin and diversity of vertebrates. In
vertebrates and other metazoans, Hox genes determine cell fate along the
anteroposterior axis of embryos and are implicated in driving morphological
diversity. Invertebrates contain a single Hox cluster (either intact or
fragmented), whereas elephant shark, coelacanth, and tetrapods contain four Hox
clusters owing to two rounds of whole-genome duplication ("1R" and "2R") during
early vertebrate evolution. By contrast, most teleost fishes contain up to eight
Hox clusters because of an additional "teleost-specific" genome duplication
event. By sequencing bacterial artificial chromosome (BAC) clones and the whole
genome, here we provide evidence for at least six Hox clusters in the Japanese
lamprey (Lethenteron japonicum). This suggests that the lamprey lineage has
experienced an additional genome duplication after 1R and 2R. The relative age
of lamprey and human paralogs supports this hypothesis. Compared with
gnathostome Hox clusters, lamprey Hox clusters are unusually large. Several
conserved noncoding elements (CNEs) were predicted in the Hox clusters of
lamprey, elephant shark, and human. Transgenic zebrafish assay indicated the
potential of CNEs to function as enhancers. Interestingly, CNEs in individual
lamprey Hox clusters are frequently conserved in multiple Hox clusters in
elephant shark and human, implying a many-to-many orthology relationship between
lamprey and gnathostome Hox clusters. Such a relationship suggests that the
first two rounds of genome duplication may have occurred independently in the
lamprey and gnathostome lineages. |
Give an overview of visualizing genomes with oligopaint FISH probes. | Oligopaint probes are fluorescently labeled, single-stranded DNA oligonucleotides that can be used to visualize genomic regions ranging in size from tens of kilobases to many megabases. Coupled with fluorescence in situ hybridization (FISH) and a bioinformatic platform, this technology could be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. The method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of chromosomal regions. It is anticipated that this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes. | A host of observations demonstrating the relationship between nuclear
architecture and processes such as gene expression have led to a number of new
technologies for interrogating chromosome positioning. Whereas some of these
technologies reconstruct intermolecular interactions, others have enhanced our
ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-
and PCR-based strategy for fluorescence in situ hybridization (FISH) and a
bioinformatic platform that enables this technology to be extended to any
organism whose genome has been sequenced. The oligonucleotide probes are
renewable, highly efficient, and able to robustly label chromosomes in cell
culture, fixed tissues, and metaphase spreads. Our method gives researchers
precise control over the sequences they target and allows for single and
multicolor imaging of regions ranging from tens of kilobases to megabases with
the same basic protocol. We anticipate this technology will lead to an enhanced
ability to visualize interphase and metaphase chromosomes. |
What is Targeted Chromatin Capture (T2C)? | Targeted Chromatin Capture (T2C) is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research. | To maintain genome function and stability, DNA sequence and its organization
into chromatin must be duplicated during cell division. Understanding how entire
chromosomes are copied remains a major challenge. Here, we use nascent chromatin
capture (NCC) to profile chromatin proteome dynamics during replication in human
cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity
purification and quantitative proteomics. Comparing nascent chromatin with
mature post-replicative chromatin, we provide association dynamics for 3,995
proteins. The replication machinery and 485 chromatin factors such as CAF-1,
DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors
including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This
correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3
and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with
experimentally derived chromatin probabilities to predict a function in nascent
chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication
factor required for PCNA loading. Together, this provides an extensive resource
to understand genome and epigenome maintece. |
What are the reported adverse effects of topical minoxidil? | Typical side effects of this topical treatment include irritative dermatitis going along with pruritus, erythema, scaling and dryness, which occur especially at the onset of the therapy. In some cases, allergic contact dermatitis or exacerbation of seborrheic dermatitis has been reported.
Hypertrichosis is a well-recognized adverse effect of therapy with either oral or topical minoxidil.
We observed an as yet unreported "polymyalgia syndrome" in four otherwise healthy males whose sole medication was topically applied minoxidil. They experienced fatigue, weight loss and severe pain in the shoulders and pelvic girdle, suggesting connective tissue disease. Three patients had a transient rise in liver enzymes, while other laboratory analyses remained normal. Tritanomaly was detected in two patients who underwent systematic color vision testing.
A case of central serous chorioretinopathy after application of topical minoxidil solution.
A case of acute myocardial infarction associated with topical use of minoxidil (RiUP) for treatment of baldness.
Compared with placebo, topical minoxidil caused significant increases in LV end-diastolic volume, in cardiac output (by 0.751 min-1) and in LV mass (by 5 g m-2).
Two of our patients developed smoking intolerance during treatment with topical minoxidil for androgenital alopecia. | Topical minoxidil, used in the treatment of baldness, has been commercially
available since 1987. Its systemic side effects are rare. We observed an as yet
unreported "polymyalgia syndrome" in four otherwise healthy males whose sole
medication was topically applied minoxidil. They experienced fatigue, weight
loss and severe pain in the shoulders and pelvic girdle, suggesting connective
tissue disease. Three patients had a transient rise in liver enzymes, while
other laboratory analyses remained normal. Tritanomaly was detected in two
patients who underwent systematic color vision testing. All symptoms disappeared
after withdrawal of minoxidil. Rechallenge was positive once in one patient and
twice in another. The mechanism of this side effect remains to be determined. Systemic cardiovascular effects during chronic treatment with topical minoxidil
vs placebo were evaluated using a double-blind, randomized design for two
parallel groups (n = 20 for minoxidil, n = 15 for placebo). During 6 months of
follow-up, blood pressure did not change, whereas minoxidil increased heart rate
by 3-5 beats min-1. Compared with placebo, topical minoxidil caused significant
increases in LV end-diastolic volume, in cardiac output (by 0.751 min-1) and in
LV mass (by 5 g m-2). We conclude that in healthy subjects short-term use of
topical minoxidil is likely not to be detrimental. However, safety needs to be
established regarding ischaemic symptoms in patients with coronary artery
disease as well as for the possible development of LV hypertrophy in healthy
subjects during years of therapy. The pathogenesis of hair loss, the postulated mechanisms of minoxidil action on
hair growth, and clinical trials, adverse reactions, experimental formulations,
and percutaneous absorption of topical minoxidil preparations are reviewed.
Topical minoxidil seems to normalize hair follicles and increase blood flow to
the scalp. In clinical trials of various formulations, results have varied.
Improved hair growth occurred after four to six months of therapy; twice-daily
application seems to be indicated. The most frequently reported adverse
reactions are mild scalp dryness and irritation and, rarely, allergic contact
dermatitis. Current recommendations are to reserve topical minoxidil for
patients with normal cardiovascular status and to routinely monitor blood
pressure, heart rate, and electrocardiographic changes. A new drug application
is pending with FDA for use of topical minoxidil in androgenetic alopecia
(male-pattern baldness), which is genetically determined and apparently
stimulated by androgens. For alopecia areata, which involves hair loss on the
body or scalp, usually patchy and of sudden onset, no reliable treatment has
been found, although minoxidil may be efficacious in some patients. Minoxidil
has generated new interest in hair-loss research. The etiology of hair loss must
be better understood before more effective treatment regimens can be designed. Eight deaths that occurred during Upjohn-sponsored clinical trials of topical
minoxidil and two deaths in subjects who used extemporaneous formulations of the
drug are summarized. Of the eight patients in clinical trials, five had
cardiovascular abnormalities and two had acquired immunodeficiency
syndrome-related pneumonia. One patient died of a self-inflicted gunshot wound.
One of the subjects who was using extemporaneous topical minoxidil had
hypertension and arteriosclerotic disease and the other died of a myocardial
infarction. There is little likelihood of significant adverse effects
attributable to topical minoxidil because of its low systemic absorption. The
evidence suggests that these deaths were the result of causes other than use of
the drug. Generalized hypertrichosis is a common side-effect of oral minoxidil treatment
for hypertension. However, hypertrichosis is uncommon after treatment with
topical minoxidil for alopecia, and normally only occurs in areas close to the
site of application. A 16-year-old girl is presented who developed generalized
hypertrichosis 3 months after applying topical minoxidil for treatment of
diffuse alopecia in doses greater than that prescribed. Four months after
discontinuing treatment, the abnormal hair gradually diminished and disappeared. A 45-year-old Japanese man with paroxysmal atrial fibrillation (AF) developed
acute anteroseptal myocardial infarction (MI). He had used 1% topical minoxidil
(RiUP) once a day for 4 months before the onset of MI for treatment of baldness.
Coronary angiography demonstrated severe stenosis at the proximal portion of the
left anterior descending coronary artery with a tilling defect.
Electrocardiographic monitoring revealed paroxysmal AF and sinus bradycardia
with sinus arrests, suggestive of sick sinus syndrome. Topical minoxidil is now
widely used for the treatment of male pattern baldness. Although it may be
difficult to relate topical use of minoxidil to myocardial ischemia, a greater
awareness of its toxicity will be necessary, and patients with cardiovascular
disorders should be excluded from the therapy. After more than a decade of use, topical minoxidil solution has proven to be a
safe and effective treatment for androgenetic alopecia. However, some patients
present with complaints of pruritus and scaling of the scalp. The most common
causes of these symptoms include irritant contact dermatitis, allergic contact
dermatitis, or an exacerbation of seborrheic dermatitis. Patients suffering from
allergic contact dermatitis may benefit from patch testing to determine the
causative allergen. Among the patients we patch tested, propylene glycol was
found to be the contactant in a majority of cases, not the minoxidil itself.
Many of these patients may be candidates for treatment with alternative
formulations using other solvents, such as butylene glycol, polysorbate, or
glycerol. Although predictive, patch testing results do not ensure that the
compounded preparations will be tolerated. Unfortunately, patients found to be
allergic to minoxidil are no longer candidates for topical treatment of their
alopecia with any preparations of minoxidil. Minoxidil is effective in inducing hair growth in patients with androgenetic
alopecia by stimulating hair follicles to undergo transition from early to late
anagen phase. However, there have been no controlled studies of topical
minoxidil in Asian women. The objective of this trial was to investigate the
efficacy of 1% topical minoxidil for androgenetic alopecia in Japanese female
patients using a double-blind controlled method. This trial included 280
Japanese female patients aged 20 years or older with androgenetic alopecia who
were administered either 1% topical minoxidil (n = 140) or placebo (n = 140) for
24 weeks. The primary efficacy variable was mean change from baseline in
non-vellus hair count/cm(2). The mean change was 8.15 in the 1% topical
minoxidil group and 2.03 in the placebo group, with a significant difference
between groups (p < 0.001) [difference: 6.12 (two-sided 95% confidence interval
(CI): 3.29-8.96)]. Secondary variables included investigators' assessments and
patients' self-assessments. As assessed by investigators, 29.2% (40/137) of the
patients had moderate or better improvement in the 1% topical minoxidil group
compared to 11.8% (16/136) in the placebo group (p < 0.001 versus placebo). The
effect on hair growth was assessed as improved or better by 36.5% (50/137) of
the patients themselves in the 1% topical minoxidil group compared to 23.5%
(32/136) in the placebo group (p = 0.019 versus placebo). The patients tolerated
treatment with 1% topical minoxidil well without significant adverse effects. BACKGROUND: 5% topical minoxidil solution has been widely used to stimulate new
hair growth and help stop hair loss in men with androgenetic alopecia (AGA).
However, it is not convenient for patients to continue applying the solution
twice daily on a regular basis. Tretinoin is known to increase the percutaneous
absorption of minoxidil and, therefore, to enhance the response of AGA to
minoxidil. For this reason, it was assumed that tretinoin would be helpful in
alleviating the inconvenience associated with the recommended twice-daily
application of minoxidil.
OBJECTIVE: To compare the efficacy and safety of therapy using a combined
solution of 5% minoxidil and 0.01% tretinoin once daily with those of the
conventional 5% topical minoxidil therapy applied twice daily in the treatment
of AGA.
METHODS: A total of 31 male patients (aged 28-45 years, mean 39.7+/-4.5) with
AGA (Hamilton-Norwood classification type III-V) were randomly assigned into two
groups, one in which 5% minoxidil was applied to the scalp twice daily and the
other in which the combined agent was applied once daily at night together with
a vehicle placebo in the morning. The efficacy parameters were: (i) changes in
total hair count, non-vellus hair count, anagen hair ratio, linear hair growth
rate, and mean hair diameter assessed by macrophotographic image analysis; and
(ii) the patient's and investigator's subjective assessments.
RESULTS: After therapy, increases in the macrophotographic variables of total
hair count and non-vellus hair count were shown in both treatment groups. There
were no statistically significant differences between the two treatment groups
with respect to changes in macrophotographic variables or scores on subjective
global assessments by patients and the investigator. The incidence of adverse
effects such as pruritus or local irritation was similar in the 5% minoxidil
group (4 of 14 subjects) and the combined agent group (5 of 15 subjects).
CONCLUSION: The efficacy and safety of combined 5% minoxidil and 0.01% tretinoin
once-daily therapy appear to be equivalent to those of conventional 5% minoxidil
twice-daily therapy for the treatment of AGA. PURPOSE: Minoxidil is one of the drugs approved for the treatment of
androgenetic alopecia. This article presents a case of central serous
chorioretinopathy after application of topical minoxidil solution.
METHODS: We examined a 37-year-old man who complained of a positive relative
scotoma, metamorphopsia and impaired dark adaptation involving the right eye.
The patient reported an 8 month history of daily topical use but denied previous
treatment with other drugs. Dilated fundus examination of right eye revealed
central swelling located over the macula. Optical coherence tomography showed
the presence of subretinal fluid. Fluorescein angiography disclosed one focal
hyperfluorescent spot in the foveal area with minimal pigmentary changes
limitated to that area. The patient was diagnosed with central serous
chorioretinopathy (CSC) potentially related to an 8 month topical minoxidil
solution administration. One month after the drug was discontinued, normal
findings were found upon reexamination. The patient reported no previous episode
of CSC.
CONCLUSION: Major systemic side effects from topical solution of minoxidil are
rare. To our knowledge, this is the first reported case of a central serous
chorioretinopathy associated with long-term use of this drug. BACKGROUND: Female pattern hair loss, or androgenic alopecia, is the most common
type of hair loss affecting women. It is characterised by progressive shortening
of the duration of the growth phase of the hair with successive hair cycles, and
progressive follicular miniaturisation with conversion of terminal to vellus
hair follicles (terminal hairs are thicker and longer, while vellus hairs are
soft, fine, and short). The frontal hair line may or may not be preserved. Hair
loss can have a serious psychological impact on people.
OBJECTIVES: To determine the effectiveness and safety of the available options
for the treatment of female pattern hair loss in women.
SEARCH METHODS: We searched the following databases up to October 2011: the
Cochrane Skin Group Specialised Register, CENTRAL in The Cochrane Library (2011,
Issue 4), MEDLINE (from 1946), EMBASE (from 1974), PsycINFO (from 1806), AMED
(from 1985), LILACS (from 1982), PubMed (from 1947), Web of Science (from 1945),
and reference lists of articles. We also searched several online trials
registries for ongoing trials.
SELECTION CRITERIA: Randomised controlled trials that assessed the effectiveness
of interventions for female pattern hair loss in women.
DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial
quality and extracted data.
MAIN RESULTS: Twenty two trials, comprising 2349 participants, were included. A
wide range of interventions were evaluated, with 10 studies investigating the
different concentrations of minoxidil. Pooled data from 4 studies indicated that
a greater proportion of participants (121/488) treated with minoxidil reported a
moderate increase in their hair regrowth when compared with placebo (64/476)
(risk ratio (RR) = 1.86, 95% confidence interval (CI) 1.42 to 2.43). In 7
studies, there was an important increase of 13.28 in total hair count per cm(2)
in the minoxidil group compared to the placebo group (95% CI 10.89 to 15.68).
There was no difference in the number of adverse events in the twice daily
minoxidil and placebo intervention groups, with the exception of a reported
increase of adverse events (additional hair growth on areas other than the
scalp) with minoxidil (5%) twice daily. Most of the other comparisons consisted
of single studies. These were assessed as high risk of bias: They did not
address our prespecified outcomes and provided limited evidence of either the
efficacy or safety of these interventions.
AUTHORS' CONCLUSIONS: Although more than half of the included studies were
assessed as being at high risk of bias, and the rest at unclear, there was
evidence to support the effectiveness and safety of topical minoxidil in the
treatment of female pattern hair loss. Further direct comparison studies of
minoxidil 5% applied once a day, which could improve adherence when compared to
minoxidil 2% twice daily, are still required. Consideration should also be given
to conducting additional well-designed, adequately-powered randomised controlled
trials investigating several of the other treatment options. Hypertrichosis is a well-recognized adverse effect of therapy with either oral
or topical minoxidil. We report a case of fronto-temporal hypertrichosis
occurring in an 8-year-old girl treated for patchy alopecia areata of the
frontal area of the scalp with 2% minoxidil solution. After failure of 5-months
minoxidil-discontinuation, hair removal with Nd:YAG laser (1064 nm line)
(Smartepil II, Deka) was tested leading to complete resolution within 2
sessions. |
Is arimoclomol a co-inducer of the heat shock response? | Yes, arimoclomol is a hydroxylamine derivative, a group of compounds which have unique properties as co-inducers of heat shock protein expression, but only under conditions of cellular stress. | Arimoclomol is an investigational drug for amyotrophic lateral sclerosis (ALS)
that amplifies heat shock protein gene expression during cell stress. The
objectives of the present study were to assess the safety, tolerability, and
pharmacokinetics of arimoclomol in ALS. Eighty-four participants with ALS
received arimoclomol at one of three oral doses (25, 50, or 100 mg three times
daily) or placebo. The primary outcome measure was safety and tolerability. A
subset of 44 participants provided serum and cerebrospinal fluid (CSF) samples
for pharmacokinetic analysis. Participants who completed 12 weeks of treatment
could enroll in a 6-month open-label study. Arimoclomol at doses up to 300
mg/day was well tolerated and safe. Arimoclomol resulted in dose-linear
pharmacologic exposures and the half-life did not change with continued
treatment. Arimoclomol CSF levels increased with dose. Arimoclomol was shown to
be safe, and it crosses the blood-brain barrier. Serum pharmacokinetic profiles
support dosing of three times per day. An efficacy study in ALS is planned. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder
characterized by motoneuron degeneration, resulting in muscle paralysis and
death, typically within 1-5 years of diagnosis. Although the pathogenesis of ALS
remains unclear, there is evidence for the involvement of proteasome dysfunction
and heat shock proteins in the disease. We have previously shown that treatment
with a co-inducer of the heat shock response called arimoclomol is effective in
the SOD(G93A) mouse model of ALS, delaying disease progression and extending the
lifespan of SOD(G93A) mice (Kieran et al. 2004). However, this previous study
only examined the effects arimoclomol when treatment was initiated in pre- or
early symptomatic stages of the disease. Clearly, to be of benefit to the
majority of ALS patients, any therapy must be effective after symptom onset. In
order to establish whether post-symptomatic treatment with arimoclomol is
effective, in this study we carried out a systematic assessment of different
treatment regimes in SOD(G93A) mice. Treatment with arimoclomol from early (75
days) or late (90 days) symptomatic stages significantly improved muscle
function. Treatment from 75 days also significantly increased the lifespan of
SOD(G93A) mice, although treatment from 90 days has no significant effect on
lifespan. The mechanism of action of arimoclomol involves potentiation of the
heat shock response, and treatment with arimoclomol increased Hsp70 expression.
Interestingly, this up-regulation in Hsp70 was accompanied by a decrease in the
number of ubiquitin-positive aggregates in the spinal cord of treated SOD(G93A)
mice, suggesting that arimoclomol directly effects protein aggregation and
degradation. Pharmacological up-regulation of heat shock proteins (hsps) rescues motoneurons
from cell death in a mouse model of amyotrophic lateral sclerosis. However, the
relationship between increased hsp expression and neuronal survival is not
straightforward. Here we examined the effects of two pharmacological agents that
induce the heat shock response via activation of HSF-1, on stressed primary
motoneurons in culture. Although both arimoclomol and celastrol induced the
expression of Hsp70, their effects on primary motoneurons in culture were
significantly different. Whereas arimoclomol had survival-promoting effects,
rescuing motoneurons from staurosporin and H(2)O(2) induced apoptosis, celastrol
not only failed to protect stressed motoneurons from apoptosis under same
experimental conditions, but was neurotoxic and induced neuronal death.
Immunostaining of celastrol-treated cultures for hsp70 and activated caspase-3
revealed that celastrol treatment activates both the heat shock response and the
apoptotic cell death cascade. These results indicate that not all agents that
activate the heat shock response will necessarily be neuroprotective. Arimoclomol, an amplifier of heat shock protein expression involved in cellular
stress response, has emerged as a potential therapeutic candidate in amyotrophic
lateral sclerosis (ALS) in recent years. Treatment with arimoclomol was reported
to improve survival and muscle function in a mouse model of motor neuron
disease. Several single- and multiple-dose safety studies have been completed in
healthy control subjects. A 3-month Phase IIa study in people with ALS
demonstrated safety at dosages up to 300 mg/day and another study is currently
recruiting participants with familial ALS caused by mutations in the superoxide
dismutase gene. We review the rationale for testing arimoclomol in sporadic and
familial ALS in the context of available safety and pharmacokinetic data.
Published and unpublished literature relative to the drug in the past two
decades is discussed. The current review attempts to bring together our existing
understanding of the actions of arimoclomol with the disease profile of ALS. The
pharmacological profile of arimoclomol and the available preclinical data make
it a promising therapeutic possibility in ALS. Recent years have seen an explosion of research into increasingly prevalent
neurodegenerative diseases. Arimoclomol (BRX-220), being developed by CytRx
Corp, is an oral therapeutic candidate for the treatment of amyotrophic lateral
sclerosis (ALS), the most common form of motor neuron disease. ALS is a fatal,
incurable disorder, which can present as sporadic (90 to 95% of cases) or
familial (5 to 10% of cases) forms. The etiology of sporadic ALS remains unknown
and much of the understanding of ALS pathogenesis has been derived through study
of its familial forms; in particular, through study of autosomal domit
mutations in the SOD1 (copper/zinc superoxide dismutase) gene, which cause
approximately 20% of familial ALS cases. Under conditions of excessive stress,
arimoclomol induces amplification of the cytoprotective heat shock response in
order to protect motor neurons from death. Comprehensive in vivo and in vitro
studies demonstrated its effect in the prevention of neuronal loss and promotion
of motor neuron survival, even after symptom onset. Clinical trials have
reported good tolerability and safety. This paper discusses the rationale for
arimoclomol use in ALS, the preclinical and clinical evidence collected to date,
the likelihood of its promising preclinical results translating to humans, and
the relevance of this research for neurodegeneration as a whole. We undertook a longitudinal study of the histological and biochemical changes at
the neuromuscular junction (NMJ) in muscles of SOD1-G93A mice. We also assessed
these functions in mice treated with a known heat shock protein inducer,
arimoclomol. Tissue samples of treated and untreated mSOD mice were analysed for
AChE and ChAT enzyme activities as markers of neuromuscular function. Sections
of hindlimb muscles (TA, EDL and soleus) were also stained for succinate
dehydrogenase and silver cholinesterase activities as well as for
immunohistochemistry. Hsp70 levels were also measured from muscle samples using
ELISA. Results showed that denervation and nerve sprouting were present at
symptom onset in fast muscles, although slow muscles remained fully innervated.
Cholinergic enzyme activities were reduced prior to denervation and declined
further with disease progression. Reduction of endplate size, a slow to fast
shift in muscle phenotype was also observed. Treatment with arimoclomol delayed
the appearance of these changes, increased innervation, cholinergic enzyme
activities and endplate size and reversed muscle fibre transformation. These
beneficial effects of arimoclomol in muscles were accompanied by an increase in
Hsp70 expression. In conclusion, our results indicate that pharmacological
targeting of muscles at early stages of disease may be a successful strategy to
ameliorate disease progression in ALS. Spinal and bulbar muscular atrophy, also known as Kennedy's disease, is an
adult-onset hereditary neurodegenerative disorder caused by an expansion of the
polyglutamine repeat in the first exon in the androgen receptor gene.
Pathologically, the disease is defined by selective loss of spinal and bulbar
motor neurons causing bulbar, facial and limb weakness. Although the precise
disease pathophysiology is largely unknown, it appears to be related to abnormal
accumulation of the pathogenic androgen receptor protein within the nucleus,
leading to disruption of cellular processes. Using a mouse model of spinal and
bulbar muscular atrophy that exhibits many of the characteristic features of the
human disease, in vivo physiological assessment of muscle function revealed that
mice with the pathogenic expansion of the androgen receptor develop a motor
deficit characterized by a reduction in muscle force, abnormal muscle
contractile characteristics, loss of functional motor units and motor neuron
degeneration. We have previously shown that treatment with arimoclomol, a
co-inducer of the heat shock stress response, delays disease progression in the
mutant superoxide dismutase 1 mouse model of amyotrophic lateral sclerosis, a
fatal motor neuron disease. We therefore evaluated the therapeutic potential of
arimoclomol in mice with spinal and bulbar muscular atrophy. Arimoclomol was
administered orally, in drinking water, from symptom onset and the effects
established at 18 months of age, a late stage of disease. Arimoclomol
significantly improved hindlimb muscle force and contractile characteristics,
rescued motor units and, importantly, improved motor neuron survival and
upregulated the expression of the vascular endothelial growth factor which
possess neurotrophic activity. These results provide evidence that upregulation
of the heat shock response by treatment with arimoclomol may have therapeutic
potential in the treatment of spinal and bulbar muscular atrophy and may also be
a possible approach for the treatment of other neurodegenerative diseases. Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness
due to the progressive death of rod and cone photoreceptors in the retina. There
are currently no effective treatments for RP. Inherited mutations in rhodopsin,
the light-sensing protein of rod photoreceptor cells, are the most common cause
of autosomal-domit RP. The majority of mutations in rhodopsin, including the
common P23H substitution, lead to protein misfolding, which is a feature in many
neurodegenerative disorders. Previous studies have shown that upregulating
molecular chaperone expression can delay disease progression in models of
neurodegeneration. Here, we have explored the potential of the heat-shock
protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of
P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved
viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat
models, pharmacological potentiation of the stress response with arimoclomol
improved electroretinogram responses and prolonged photoreceptor survival, as
assessed by measuring outer nuclear layer thickness in the retina. Furthermore,
treated animal retinae showed improved photoreceptor outer segment structure and
reduced rhodopsin aggregation compared with vehicle-treated controls. The
heat-shock response (HSR) was activated in P23H retinae, and this was enhanced
with arimoclomol treatment. Furthermore, the unfolded protein response (UPR),
which is induced in P23H transgenic rats, was also enhanced in the retinae of
arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR
as well as the HSR. These data suggest that pharmacological enhancement of
cellular stress responses may be a potential treatment for rhodopsin RP and that
arimoclomol could benefit diseases where ER stress is a factor. |
What memory problems are reported in the " Gulf war syndrome" | memory loss | BACKGROUND: Numerous questions have been raised about the health consequences to
veterans of the Gulf War but most particularly to issues concerning women, who
were deployed in unprecedented numbers. Little is known about the health
consequences to women of wartime stressors, in general, or the environmental and
job-related exposures specific to the theater of the Gulf War.
METHODS: A stratified sample of 525 women participated in the study following
the war and again in a follow-up study 2 yr later. The sampling frame was
stratified on component of the U.S. Air Force (active, guard or reserve),
deployment (in the theater or elsewhere), and parental status (parent or
nonparent). Measures included items concerning general physical health,
gender-specific health, the "Gulf War Syndrome," and the emotional responses to
war, including symptoms of post-traumatic stress disorder (PTSD).
RESULTS: Multiple statistical analyses were used to describe women's physical
and emotional health at two time points following the war. Women deployed to the
theater reported significantly more general as well as gender-specific health
problems than did women deployed elsewhere. A cluster of common health problems
included: skin rash, cough, depression, unintentional weight loss, insomnia, and
memory problems. Women serving in the theater also reported a significant
increase in several gender-specific problems compared to women deployed
elsewhere.
CONCLUSIONS: Findings suggest the need for follow-up of a cluster of specific
health effects, including those concerning gynecologic and reproductive health. The Gulf War syndrome represents neurological and neuropsychological disorders
in veterans of the Persian Gulf war. Until today, the various symptoms observed
could not be attributed to any defined disease. As a possible cause, exposure to
neurotoxic agents such as the organophosphates used during the war has been
suggested by many authors. We report on a 29-year-old man who suffered from
dysmnesia, disturbance of orientation, cognitive impairment, and double vision.
His history revealed several front-line operations in 1990 and 1991 during the
Gulf War. Physical examinations showed a complex eye-movement disturbance and a
horizontal nystagmus, which was neuro-ophthalmologically confirmed. The early
auditory potentials referred to a brainstem dysfunction and the cognitive
disturbances correlated to changes in the late-appearing component of
event-related potentials (P 300). Brain imaging with CCT, MRI, SPECT, PET, and
EEG and CSF showed no pathologies. Neuropsychological tests disclosed severe
cognitive impairment especially concerning memory. Three-month follow-up studies
in a department of psychosomatic medicine excluded a dissociative disorder as a
feature of a post-traumatic stress or a conversion disorder. This is the first
case of Gulf War syndrome in Germany. We focus on an unfamiliar complication
after the war. The recent literature is reviewed. In early 1992, U.S. troops returning from the Gulf War began reporting a variety
of nonspecific symptoms such as fatigue, skin rash, headache, muscle and joint
pain, and loss of memory. These reports marked the beginning of what was to be
identified as the Gulf War Syndrome (GWS). In the years since the war, as many
as 100,000 troops have claimed they suffer from this mysterious disease. In our
culture, the existence of disease as a specific entity is fundamental to
ensuring the validity of that disease. The legitimacy of GWS has been repeatedly
called into question because no specific physiological etiology has been
confirmed, and it is becoming more and more likely that the origin of GWS will
never be clearly delineated. The purpose of this paper is to illustrate the
complicated process of defining GWS as a legitimate illness in the absence of
etiological evidence and to suggest a method of treatment for individuals who
still suffer from its sequelae. BACKGROUND: Forces deployed to the first Gulf War report more ill health than
veterans who did not serve there. Many studies of post-Gulf morbidity are based
on relatively small sample sizes and selection bias is often a concern. In a
setting where selection bias relating to the ill health of veterans may be
reduced, we: i) examined self-reported adult ill health in a large sample of
male UK Gulf War veterans and a demographically similar non-deployed comparison
group; and ii) explored self-reported ill health among veterans who believed
that they had Gulf War syndrome.
METHODS: This study uses data from a retrospective cohort study of reproduction
and child health in which a validated postal questionnaire was sent to all UK
Gulf War veterans (GWV) and a comparison cohort of Armed Service personnel who
were not deployed to the Gulf (NGWV). The cohort for analysis comprises 42,818
males who responded to the questionnaire.
RESULTS: We confirmed that GWV report higher rates of general ill health. GWV
were significantly more likely to have reported at least one new medical symptom
or disease since 1990 than NGWV (61% versus 37%, OR 2.7, 95% CI 2.5-2.8). They
were also more likely to report higher numbers of symptoms. The strongest
associations were for mood swings (OR 20.9, 95%CI 16.2-27.0), memory loss/lack
of concentration (OR 19.6, 95% CI 15.5-24.8), night sweats (OR 9.9, 95% CI
6.5-15.2), general fatigue (OR 9.6, 95% CI 8.3-11.1) and sexual dysfunction (OR
4.6, 95%CI 3.2-6.6). 6% of GWV believed they had Gulf War syndrome (GWS), and
this was associated with the highest symptom reporting.
CONCLUSIONS: Increased levels of reported ill health among GWV were confirmed.
This study was the first to use a questionnaire which did not focus specifically
on the veterans' symptoms themselves. Nevertheless, the results are consistent
with those of other studies of post-Gulf war illness and thus strengthen overall
findings in this area of research. Further examination of the mechanisms
underlying the reporting of ill health is required. |
Describe Heyde syndrome. | Classical Heyde syndrome is described as the association of aortic stenosis, bleeding gastrointestinal angiodysplasia and secondary anemia. A deficiency of high molecular weight multimers of von Willebrand factor (type 2A von Willebrand disease) provides the link between this association. | The association between aortic stenosis and digestive angiodysplasia has been
described for the first time by Heyde in 1958. This entity is thus known as
Heyde's syndrome. In many instances, the recurrent small intestinal bleeding
originating from angiodysplasia stopped after aortic valve replacement. We
report two cases of patients presenting with a recurrent small intestinal
bleeding originating from digestive angiodysplasia and suffering from aortic
stenosis. Diagnosis of both pathologies is well documented in both cases. The
replacement of the aortic valve by a biologic prosthesis stopped the bleeding.
Prior to aortic valve replacement, the two patients suffered severe recurrent
blood loss from intestinal angiodysplasia. The treatment of aortic stenosis
greatly favored both cardiac and general status. We recommend aortic valve
replacement with a biologic prosthesis prior to intestinal resection in patients
presenting with Heyde's syndrome. We stress on the fact that anticoagulants must
be stopped in order to minimize the risk of further bleeding. The coincidence of aortic valve stenosis and cryptogenic gastrointestinal
bleeding usually from angiodysplasia of the cecum and ascending colon has been
called Heyde syndrome. The pathophysiologic link between both remains unclear
and may be due to subtle changes in plasmatic coagulation. There are some
statistic and numerous casuistic reports, and recently the existence of the
syndrome has been questioned. We describe two cases of aortic valve stenosis and
cryogenic gastrointestinal bleeding in which bleeding subsided after replacement
with a bioprosthesis. We give an overview of the current literature. BACKGROUND: The Heyde syndrome describes the coincidence of aortic valve
stenosis and intestinal bleeding. The pathophysiologic link between both
entities has remained unclear so far. In several studies the intestinal
bleedings have been attributed to angiodysplasia. Cessations of the intestinal
bleedings following replacement of the aortic valve have been described by
numerous reports.
CASE REPORT: We describe the history of a 70-year-old man with a calcified
aortic valve stenosis. In addition, he suffered from recurrent severe epistaxis
over several years. Replacement of the aortic valve by bioprosthesis resulted in
complete cessation of the nasal bleedings over a follow-up period of 6 years.
CONCLUSION: This case report suggests that aortic valve stenoses may be
associated with extraintestinal bleedings as well. BACKGROUND: Heyde syndrome is described as the association of arteriovenous
malformations (AVMs) of the gastrointestinal tract and aortic stenosis (AS); its
existence, however, has been questioned. We examined whether there is an
association between AVMs and AS when objective measures are used to diagnose
these findings.
METHODS: We identified all patients who were diagnosed with AVMs between 1990
and 2000 by means of gastrointestinal endoscopy or mesenteric angiography. We
compared the prevalence of AS and mitral stenosis (MS) in the 73 patients with
AVMs who also had echocardiograms. For a comparison with the general population,
the prevalence of AS and MS in all patients who had echocardiograms between 1990
and 2000 was calculated (MS was chosen for comparison as a lesion with similar
likelihood of prompting an echocardiographic evaluation).
RESULTS: The prevalence of AS was 31.7% in patients with AVMs, which was
significantly higher than the 14.0% found in the general population comparison
group (P<.001). The prevalence of MS was 1.6% in the AVM group, which was not
statistically different from the 6.0% MS prevalence in the general
echocardiogram population (P =.14). Significant AS was 2.6 times more common,
and severe AS was 4.1 times more common, in patients with AVMs than in the
general population. Age and sex were not associated with Heyde syndrome, but the
association was more prevalent in blacks.
CONCLUSIONS: Our study confirmed an association between AVMs and AS, although
the etiology of the Heyde syndrome remains unclear. Clinicians need to be aware
of this syndrome because it may affect their management of patients with
gastrointestinal bleeding or AS. The authors report a case of small bowel bleeding diagnosed by Tc-99m-labeled
red blood cell (RBC) scintigraphy during the postoperative period after aortic
valve replacement. There is a relationship between aortic valve stenosis and
gastrointestinal bleeding in elderly patients, called Heyde syndrome. The
described patient had chronic anemia that worsened after surgery. RBC
scintigraphy localized the source of bleeding from jejunal angiodysplasia
confirmed by mesenteric angiography. This case illustrates the diagnostic
information provided by RBC scintigraphy in this syndrome. Angiodysplasia are common in patients over the age of 60. Heyde syndrome
describes the coincidence of aortic valve stenosis and gastrointestinal bleeding
from angiodysplasia. We describe one characteristic case of aortic valve
stenosis and gastrointestinal bleeding from angiodysplasia which subsided after
replacement with an aortic valve bioprosthesis. We review the current literature
and discuss the actual explanation approaches for this phenomenon.
CONCLUSION: There seems to be a clear indication for valve replacement in the
case of aortic valve-stenosis and gastrointestinal bleeding due to
angiodysplasia. Heyde syndrome is a triad of aortic stenosis, an acquired coagulopathy and
anaemia due to bleeding from intestinal angiodysplasia. The evidence that aortic
stenosis is the root cause of this coagulopathy is compelling. Resolution of
anaemia usually follows aortic valve replacement. This article discusses studies
linking aortic stenosis with other conditions in the triad as well as diagnosis
and management of this complex pathology. Heyde's syndrome was first proposed in 1958. It refers to gastrointestinal
haemorrhage resulting from a combination of aortic stenosis with angiodysplasia.
This report explores the case of a 93-year-old lady who was admitted to hospital
following a neck of femur fracture. She suffered from multiple comorbidities
including renal failure and congestive heart failure secondary to critical
aortic stenosis. As an inpatient she suffered an exacerbation of both her heart
and renal failure postoperatively. A week later she suffered from heavy upper
gastro-intestinal bleeding, which failed to respond to pharmacological and
endoscopic therapies as well as angiographic embolisation. The pathophysiology
of Heyde's syndrome: an acquired von Willebrand deficiency syndrome has a much
wider impact than was commonly thought, both in terms of how common it is and in
how the association may be extrapolated to a wide range of bleeding disorders,
rather than simply angiodysplasia associated gastrointestinal haemorrhage. We report the case of a 71-year-old man with diagnosis of aortic valve stenosis
for ten years, who came to hospital because of breathlessness during the
previous two months and recent low intestinal hemorrhage. On admission,
laboratory tests and upper gastrointestinal endoscopy and colonoscopy revealed
anemia and bleeding cecal angiodysplasia. The echocardiography study showed a
severe aortic stenosis. Classical Heyde syndrome is described as the association
of aortic stenosis, bleeding gastrointestinal angiodysplasia and secondary
anemia. The antecedent of mediastinal radiotherapy for treatment of Hodgkin's
disease during his youth, and eventual late cardiac adverse effects that may
include aortic or mitral valve disturbances are highlighted. Electrocoagulation
with argonium was performed on the sites of active bleeding during the
colonoscopy. In sequence, surgical replacement by bioprothesis was done on the
aortic valve. The patient remains asymptomatic, under long-term outpatient
surveillance, with normal control evaluations. The aim of this case study is to
emphasize difficulties related to diagnosis, and to highlight the role of
endoscopy and imaging studies to confirm a hypothesis of this underestimated
condition. The association between aortic valve stenosis and gastrointestinal bleeding,
traditionally known as Heyde's syndrome, is the result of a quantitative loss of
the highest molecular weight von Willebrand multimers (type 2A von Willebrand
syndrome). This results in bleeding from areas of high shear stress such as
gastrointestinal angiodysplasias. Correction of this bleeding diathesis after
surgical aortic valve replacement has been well described. The effect of
transcutaneous aortic valve implantation on Heyde's syndrome has yet to be
studied. Herein, we report a patient with severe aortic stenosis, type 2A von
Willebrand syndrome, and hemorrhagic shock from gastrointestinal bleeding who
underwent successful transcutaneous aortic valve implantation. BACKGROUND: Mitral valve regurgitation is associated with an acquired hemostatic
defect.
OBJECTIVE: We sought to assess the prevalence and severity of acquired von
Willebrand syndrome in patients with native valve mitral regurgitation (MR).
PATIENTS/METHODS: Fifty-three patients were prospectively observed with bleeding
questionnaires and laboratory tests when undergoing an echocardiographic
assessment of MR. In patients referred for mitral valve surgery, testing was
repeated postoperatively.
RESULTS: Echocardiography identified 13 patients with mild MR, 14 with moderate
MR, and 26 with severe MR. Among patients with mild, moderate or severe MR, loss
of the highest molecular weight von Willebrand factor (VWF) multimers occurred
in 8%, 64%, and 85%, respectively, median platelet function analyzer collagen
ADP closure times (PFA-CADPs) were 84 s (interquartile range [IQR] 73-96 s), 156
s (IQR 104-181 s), and 190 s (IQR 157-279 s), respectively, and the ratios of
VWF latex activity to antigen were 0.92 (IQR 0.83-0.97), 0.85 (IQR 0.76-0.89),
and 0.79 (IQR 0.75-0.82), respectively (all P < 0.001). Nine patients reported
clinically significant bleeding, and seven had intestinal angiodysplasia and
transfusion-dependent gastrointestinal bleeding (Heyde syndrome), with the
median number of transfusions required being 20 (IQR 10-33; range 4-50). In
patients who underwent mitral valve repair (n = 13) or replacement (n = 7), all
measures of VWF function reported above improved significantly.
CONCLUSION: The high-shear environment of moderate to severe MR is sufficient to
produce prevalent perturbations in VWF activity. Acquired von Willebrand
syndrome may occur in this setting, and appears to be reversible with mitral
valve surgery. Heyde syndrome is a triad of aortic stenosis, acquired coagulopathy, and anemia
due to bleeding from intestinal angiodysplasia. Here we describe a case of this
syndrome. An 80-year-old woman with severe aortic stenosis was referred to our
department for an aortic valve replacement. She suffered from recurrent
iron-deficiency anemia and required transfusions every 2 weeks. Gastroscopy and
colonoscopy were normal with the exception of angiodysplasia without bleeding in
the cecum. After aortic valve replacement her anemia was resolved. She was
discharged on postoperative day 22. No transfusions were needed after the
procedure. To date, her hemoglobin has remained stable at >10 mg/dL. |
Has silicon been used in treatment of incontinence ? | Yes | Objective: This study was undertaken in order to determine if the "Femassist"
device is a safe and effective treatment for women with the diagnosis of urinary
incontinence.Methods: The Femassist is a medical-grade silicon dome-shaped
device, worn over the urethra and held securely via suction and a commercially
available adhesive lotion. Women with a chief complaint of urinary incontinence
responding to local newspaper advertisements were screened for inclusion.
Potential candidates underwent medical history, physical (including gynecologic)
examination, Papanicolaou test, urine culture and cytology, and multichannel
urodynamic testing (including abdominal leak-point pressure measurements). A
total of 38 women with documented genuine stress urinary incontinence (GSUI) or
mixed incontinence were ultimately recruited into the study and fitted with
either the standard or petite-sized Femassist device, according to their
individual anatomy. Subjects were assessed before and after 1 month's use.
Subjective assessment included quality of life questionnaires, daily voiding and
activity diaries, as well as ongoing patient comments retrieved through daily
telephone contact with the study nurse. Objective assessment included blinded
evaluation of bacteriuria and urinary infection rates and vulvar irritation and
ulceration rates.Results: To date, of the 38 women who have completed the study,
over 50% reported an improvement in their quality of life including comfort,
convenience, and overall satisfaction. In total for all patients studied, the
device was worn for a total of 886 days; 82% of these were dry days. Similar
results were obtained for women with GSUI and mixed incontinence. Factors
associated with successful experience with the device included degree of tissue
estrogenization (either naturally or via a topical estrogen preparation), manual
dexterity, and degree of motivation. One in five women reported vulvar
irritation or urethral discomfort at some point; this was not correlated with
percentage of dry days. There have been no reported significant increases in
bacteriuria or urinary tract infection rates over patients' baseline
experiences.Conclusion: Our preliminary study suggests that the Femassist device
is a safe and effective method for the management of female urinary
incontinence. OBJECTIVES: Stress urinary incontinence alter radical prostatectomy is one of
the most worrisome sequelae for the patient and urologist. The aim of this paper
is to evaluate the indications of the suburethral mesh Invance, giving details
on our preoperative evaluation and indication, surgical technique, and the
correlation between preoperative findings and functional results.
METHODS: Between February 2006 and January 2009 27 patients underwent surgery.
All of them had more than one year of follow up after prostatectomy, urodynamic
study and preoperative cystoscopy. Continence was evaluated by the number of
pads/day and the ICIQ-UI SF questionnaire. Through a perineal incision three
titanium screws with a polipropylene suture were inserted in each ischiopubic
rami, and a silicon/polipropylene mesh (Invance) is affixed to them, compressing
the bulbar urethra. Patients were divided into two groups: good prognosis (1-2
pads/day without urodynamic anomalies in the filling phase) and bad prognosis (3
pads/day, history of radiotherapy or bladder neck incision, and urodynamic
anomalies). Cure was defined as a patient not needing pads, and improvement was
defined as decrease in the number of pads per day.
RESULTS: Median follow up after Invance was 18 months (4-38). Nine patients used
one pad/day, 10 used two, and eight used three pads /day. Six cases had
underwent previous bladder neck incision and three radiotherapy. Globally, 20
patients (74% ) were cured and five (19% ) had improved. Cure rate was 100% in
the good prognosis group and 61% in the bad prognosis group (p=0.03). No
intraoperative complications were registered. During the immediate postoperative
period,one patient required cystostomy tube for 10 days. Seven patients (26% )
presented perineal discomfort; neither de novo urgency nor urethral erosion nor
sling explants due to infection were registered.
CONCLUSIONS: Invance is a safe technique that guarantees a high continence rate
in selected patients. It is a reproducible operation and, with a midterm follow
up, no loss of continence has been observed, and the risk of urethral erosion is
null in our experience. Patients with mild-moderate incontinence, without
urodynamic anomalies nor previous radiotherapy are the ideal candidates. This report describes creation of a bone-anchored sling with the InVance™ system
(American Medical Systems, Minnetonka, MN, USA) for the treatment of 2 patients
with incontinence after radical prostatectomy. The InVance™ system uses a
silicon-coated polyester sling positioned under the bulbar urethra via a
perineal incision. The sling is attached to both ischiopubic rami by 3 titanium
screws. Operative times were 157 minutes (patient 1) and 240 minutes (patient
2). Blood loss was 70 mL (patient 1) and 10 mL (patient 2). The patients used 7
and 5 absorbent pads/day, respectively, before surgery and 1 and 0 pads/day
after surgery (this datum does not appear in the main text, although the absence
of incontinence is mentioned). The only major adverse event encountered was mesh
infection necessitating mesh removal in patient 2. This operation appears
comparatively simple and useful. |
Which proteins compose the error prevention GO (8-oxo-G) system in Pseudomonas putida? | In P. putida (Pseudomonas putida) the error prevention GO (8-oxo-G) system is composed of MutY, MutM, and MutT enzymes. | |
List five applications of machine learning algorithms in medical diagnosis. | Machine learning technology is well suited for the induction of diagnostic and prognostic rules and solving of small and specialized diagnostic and prognostic problems. The medical diagnostic knowledge can be automatically derived from the description of cases solved in the past. In several medical domains we actually applied machine learning algorithms. Typically, the automatically generated diagnostic rules achieved the same or slightly better diagnostic accuracy than physicians specialists. There many several application of machine learning algorithms in medical diagnosis such as Brain glioma progression, Microarray classification, Mass spectral proteomics, Lymph disease classification and Parkinson's disease. | BACKGROUND: The use of mass spectrometry as a proteomics tool is poised to
revolutionize early disease diagnosis and biomarker identification.
Unfortunately, before standard supervised classification algorithms can be
employed, the "curse of dimensionality" needs to be solved. Due to the sheer
amount of information contained within the mass spectra, most standard machine
learning techniques cannot be directly applied. Instead, feature selection
techniques are used to first reduce the dimensionality of the input space and
thus enable the subsequent use of classification algorithms. This paper examines
feature selection techniques for proteomic mass spectrometry.
RESULTS: This study examines the performance of the nearest centroid classifier
coupled with the following feature selection algorithms. Student-t test,
Kolmogorov-Smirnov test, and the P-test are univariate statistics used for
filter-based feature ranking. From the wrapper approaches we tested sequential
forward selection and a modified version of sequential backward selection.
Embedded approaches included shrunken nearest centroid and a novel version of
boosting based feature selection we developed. In addition, we tested several
dimensionality reduction approaches, namely principal component analysis and
principal component analysis coupled with linear discrimit analysis. To
fairly assess each algorithm, evaluation was done using stratified cross
validation with an internal leave-one-out cross-validation loop for automated
feature selection. Comprehensive experiments, conducted on five popular cancer
data sets, revealed that the less advocated sequential forward selection and
boosted feature selection algorithms produce the most consistent results across
all data sets. In contrast, the state-of-the-art performance reported on
isolated data sets for several of the studied algorithms, does not hold across
all data sets.
CONCLUSION: This study tested a number of popular feature selection methods
using the nearest centroid classifier and found that several reportedly
state-of-the-art algorithms in fact perform rather poorly when tested via
stratified cross-validation. The revealed inconsistencies provide clear evidence
that algorithm evaluation should be performed on several data sets using a
consistent (i.e., non-randomized, stratified) cross-validation procedure in
order for the conclusions to be statistically sound. Increasing frame rate is a challenging issue for better interpretation of
medical images and diagnosis based on tracking the small transient motions of
myocardium and valves in real time visualization. In this paper, manifold
learning algorithm is applied to extract the nonlinear embedded information
about echocardiography images from the consecutive images in two dimensional
manifold spaces. In this method, we presume that the dimensionality of
echocardiography images obtained from a patient is artificially high and the
images can be described as functions of only a few underlying parameters such as
periodic motion due to heartbeat. By this approach, each image is projected as a
point on the reconstructed manifold; hence, the relationship between images in
the new domain can be obtained according to periodicity of the heart cycle. To
have a better tracking of the echocardiography, images during the fast motions
of heart we have rearranged the similar frames of consecutive heart cycles in a
sequence. This provides a full view slow motion of heart movement through
increasing the frame rate to three times the traditional ultrasound systems. Breast lesion segmentation in magnetic resoce (MR) images is one of the most
important parts of clinical diagnostic tools. Pixel classification methods have
been frequently used in image segmentation with two supervised and unsupervised
approaches up to now. Supervised segmentation methods lead to high accuracy, but
they need a large amount of labeled data, which is hard, expensive, and slow to
be obtained. On the other hand, unsupervised segmentation methods need no prior
knowledge and lead to low performance. However, semi-supervised learning which
uses not only a few labeled data, but also a large amount of unlabeled data
promises higher accuracy with less effort. In this paper, we propose a new
interactive semi-supervised approach to segmentation of suspicious lesions in
breast MRI. Using a suitable classifier in this approach has an important role
in its performance; in this paper, we present a semi-supervised algorithm
improved self-training (IMPST) which is an improved version of self-training
method and increase segmentation accuracy. Experimental results show that
performance of segmentation in this approach is higher than supervised and
unsupervised methods such as K nearest neighbors, Bayesian, Support Vector
Machine, and Fuzzy c-Means. BACKGROUND: Professionals in the biomedical domain are confronted with an
increasing mass of data. Developing methods to assist professional end users in
the field of Knowledge Discovery to identify, extract, visualize and understand
useful information from these huge amounts of data is a huge challenge. However,
there are so many diverse methods and methodologies available, that for
biomedical researchers who are inexperienced in the use of even relatively
popular knowledge discovery methods, it can be very difficult to select the most
appropriate method for their particular research problem.
RESULTS: A web application, called KNODWAT (KNOwledge Discovery With Advanced
Techniques) has been developed, using Java on Spring framework 3.1. and
following a user-centered approach. The software runs on Java 1.6 and above and
requires a web server such as Apache Tomcat and a database server such as the
MySQL Server. For frontend functionality and styling, Twitter Bootstrap was used
as well as jQuery for interactive user interface operations.
CONCLUSIONS: The framework presented is user-centric, highly extensible and
flexible. Since it enables methods for testing using existing data to assess
suitability and performance, it is especially suitable for inexperienced
biomedical researchers, new to the field of knowledge discovery and data mining.
For testing purposes two algorithms, CART and C4.5 were implemented using the
WEKA data mining framework. Accurate and effective diagnosis of actual injury severity can be problematic in
trauma patients. Inherent physiologic compensatory mechanisms may prevent
accurate diagnosis and mask true severity in many circumstances. The objective
of this project was the development and validation of a multiparameter machine
learning algorithm and system capable of predicting the need for life-saving
interventions (LSIs) in trauma patients. Statistics based on means, slopes, and
maxima of various vital sign measurements corresponding to 79 trauma patient
records generated over 110,000 feature sets, which were used to develop, train,
and implement the system. Comparisons among several machine learning models
proved that a multilayer perceptron would best implement the algorithm in a
hybrid system consisting of a machine learning component and basic detection
rules. Additionally, 295,994 feature sets from 82 h of trauma patient data
showed that the system can obtain 89.8 % accuracy within 5 min of recorded LSIs.
Use of machine learning technologies combined with basic detection rules
provides a potential approach for accurately assessing the need for LSIs in
trauma patients. The performance of this system demonstrates that machine
learning technology can be implemented in a real-time fashion and potentially
used in a critical care environment. BACKGROUND: The abundance of gene expression microarray data has led to the
development of machine learning algorithms applicable for tackling disease
diagnosis, disease prognosis, and treatment selection problems. However, these
algorithms often produce classifiers with weaknesses in terms of accuracy,
robustness, and interpretability. This paper introduces fuzzy support vector
machine which is a learning algorithm based on combination of fuzzy classifiers
and kernel machines for microarray classification.
RESULTS: Experimental results on public leukemia, prostate, and colon cancer
datasets show that fuzzy support vector machine applied in combination with
filter or wrapper feature selection methods develops a robust model with higher
accuracy than the conventional microarray classification models such as support
vector machine, artificial neural network, decision trees, k nearest neighbors,
and diagonal linear discrimit analysis. Furthermore, the interpretable
rule-base inferred from fuzzy support vector machine helps extracting biological
knowledge from microarray data.
CONCLUSIONS: Fuzzy support vector machine as a new classification model with
high generalization power, robustness, and good interpretability seems to be a
promising tool for gene expression microarray classification. BACKGROUND: Supervised machine learning has been proposed as a revolutionary
approach for identifying sensitive medical image biomarkers (or combination of
them) allowing for automatic diagnosis of individual subjects. The aim of this
work was to assess the feasibility of a supervised machine learning algorithm
for the assisted diagnosis of patients with clinically diagnosed Parkinson's
disease (PD) and Progressive Supranuclear Palsy (PSP).
METHOD: Morphological T1-weighted Magnetic Resoce Images (MRIs) of PD
patients (28), PSP patients (28) and healthy control subjects (28) were used by
a supervised machine learning algorithm based on the combination of Principal
Components Analysis as feature extraction technique and on Support Vector
Machines as classification algorithm. The algorithm was able to obtain
voxel-based morphological biomarkers of PD and PSP.
RESULTS: The algorithm allowed individual diagnosis of PD versus controls, PSP
versus controls and PSP versus PD with an Accuracy, Specificity and
Sensitivity>90%. Voxels influencing classification between PD and PSP patients
involved midbrain, pons, corpus callosum and thalamus, four critical regions
known to be strongly involved in the pathophysiological mechanisms of PSP.
COMPARISON WITH EXISTING METHODS: Classification accuracy of individual PSP
patients was consistent with previous manual morphological metrics and with
other supervised machine learning application to MRI data, whereas accuracy in
the detection of individual PD patients was significantly higher with our
classification method.
CONCLUSIONS: The algorithm provides excellent discrimination of PD patients from
PSP patients at an individual level, thus encouraging the application of
computer-based diagnosis in clinical practice. Machine learning-based classification techniques provide support for the
decision-making process in many areas of health care, including diagnosis,
prognosis, screening, etc. Feature selection (FS) is expected to improve
classification performance, particularly in situations characterized by the high
data dimensionality problem caused by relatively few training examples compared
to a large number of measured features. In this paper, a random forest
classifier (RFC) approach is proposed to diagnose lymph diseases. Focusing on
feature selection, the first stage of the proposed system aims at constructing
diverse feature selection algorithms such as genetic algorithm (GA), Principal
Component Analysis (PCA), Relief-F, Fisher, Sequential Forward Floating Search
(SFFS) and the Sequential Backward Floating Search (SBFS) for reducing the
dimension of lymph diseases dataset. Switching from feature selection to model
construction, in the second stage, the obtained feature subsets are fed into the
RFC for efficient classification. It was observed that GA-RFC achieved the
highest classification accuracy of 92.2%. The dimension of input feature space
is reduced from eighteen to six features by using GA. Mass spectrometry based proteomics technologies have allowed for a great
progress in identifying disease biomarkers for clinical diagnosis and prognosis.
However, they face acute challenges from a data reproducibility standpoint, in
that no two independent studies have been found to produce the same proteomic
patterns. Such reproducibility issues cause the identified biomarker patterns to
lose repeatability and prevent real clinical usage. In this work, we propose a
profile biomarker approach to overcome this problem from a machine-learning
viewpoint by developing a novel derivative component analysis (DCA). As an
implicit feature selection algorithm, derivative component analysis enables the
separation of true signals from red herrings by capturing subtle data behaviors
and removing system noises from a proteomic profile. We further demonstrate its
advantages in disease diagnosis by viewing input data as a profile biomarker.
The results from our profile biomarker diagnosis suggest an effective solution
to overcoming proteomics data's reproducibility problem, present an alternative
method for biomarker discovery in proteomics, and provide a good candidate for
clinical proteomic diagnosis. OBJECTIVES: The ability to differentiate between brain tumor progression and
radiation therapy induced necrosis is critical for appropriate patient
management. In order to improve the differential diagnosis, we combined
fluorine-18 2-fluoro-deoxyglucose positron emission tomography ((18)F-FDG PET),
proton magnetic resoce spectroscopy ((1)H MRS) and histological data to
develop a multi-parametric machine-learning model.
METHODS: We enrolled twelve post-therapy patients with grade 2 and 3 gliomas
that were suspicious of tumor progression. All patients underwent (18)F-FDG PET
and (1)H MRS. Maximal standardized uptake value (SUVmax) of the tumors and
reference regions were obtained. Multiple 2D maps of choline (Cho), creatine
(Cr), and N-acetylaspartate (NAA) of the tumors were generated. A support vector
machine (SVM) learning model was established to take imaging biomarkers and
histological data as input vectors. A combination of clinical follow-up and
multiple sequential MRI studies served as the basis for assessing the clinical
outcome. All vector combinations were evaluated for diagnostic accuracy and
cross validation. The optimal cutoff value of individual parameters was
calculated using Receiver operating characteristic (ROC) plots.
RESULTS: The SVM and ROC analyses both demonstrated that SUVmax of the lesion
was the most significant single diagnostic parameter (75% accuracy) followed by
Cho concentration (67% accuracy). SVM analysis of all paired parameters showed
SUVmax and Cho concentration in combination could achieve 83% accuracy. SUVmax
of the lesion paired with SUVmax of the white matter as well as the tumor Cho
paired with the tumor Cr both showed 83% accuracy. These were the most
significant paired diagnostic parameters of either modality. Combining all four
parameters did not improve the results. However, addition of two more
parameters, Cho and Cr of brain parenchyma contralateral to the tumor, increased
the accuracy to 92%.
CONCLUSION: This study suggests that SVM models may improve detection of glioma
progression more accurately than single parametric imaging methods.
RESEARCH SUPPORT: National Cancer Institute, Cancer Center Support Grant
Supplement Award, Imaging Response Assessment Teams. |
Does the 3D structure of the genome remain stable during cell differentiation? | Many studies have suggested a link between the spatial organization of genomes and fundamental biological processes such as genome reprogramming, gene expression, and differentiation. The open chromatin of embryonic stem cells (ESCs) condenses into repressive heterochromatin as cells exit the pluripotent state. The relation between alterations in chromatin structure and changes in gene expression during cell differentiation has served as a paradigm to understand the link between genome organization and function. Insulators are involved in 3D genome organization at multiple spatial scales and are important for dynamic reorganization of chromatin structure during reprogramming and differentiation. Architectural proteins orchestrate higher-order chromatin organization through the establishment of interactions between regulatory elements across multiple spatial scales. The regulation of these proteins, their interaction with DNA, and their co-occurrence in the genome, may be responsible for the plasticity of 3D chromatin architecture that dictates cell and time-specific blueprints of gene expression. | 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. Cells face the challenge of storing two meters of DNA in the three-dimensional
(3D) space of the nucleus that spans only a few microns. The nuclear
organization that is required to overcome this challenge must allow for the
accessibility of the gene regulatory machinery to the DNA and, in the case of
embryonic stem cells (ESCs), for the transcriptional and epigenetic changes that
accompany differentiation. Recent technological advances have allowed for the
mapping of genome organization at an unprecedented resolution and scale. These
breakthroughs have led to a deluge of new data, and a sophisticated
understanding of the relationship between gene regulation and 3D genome
organization is beginning to form. In this review we summarize some of the
recent findings illuminating the 3D structure of the eukaryotic genome, as well
as the relationship between genome topology and function from the level of whole
chromosomes to enhancer-promoter loops with a focus on features affecting genome
organization in ESCs and changes in nuclear organization during differentiation. Many studies have suggested a link between the spatial organization of genomes
and fundamental biological processes such as genome reprogramming, gene
expression, and differentiation. Multicolor fluorescence in situ hybridization
on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal
microscopy, has become an effective technique for analyzing 3D genome structure
and spatial patterns of defined nucleus targets including entire chromosome
territories and single gene loci. This technique usually requires the
simultaneous visualization of numerous targets labeled with different colored
fluorochromes. Thus, the number of channels and lasers must be sufficient for
the commonly used labeling scheme of 3D-FISH, "one probe-one target". However,
these channels and lasers are usually restricted by a given microscope system.
This paper presents a method for simultaneously delineating multiple targets in
3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other
labeling schemes, this method is convenient and simple for multicolor 3D-FISH
studies, which may result in widespread adoption of the technique. Lastly, as an
application of the method, the nucleus locations of chromosome territory 18/21
and centromere 18/21/13 in normal human lymphocytes were analyzed, which might
present evidence of a radial higher order chromatin arrangement. It can be convenient to think of the genome as simply a string of nucleotides,
the linear order of which encodes an organism's genetic blueprint. However, the
genome does not exist as a linear entity within cells where this blueprint is
actually utilized. Inside the nucleus, the genome is organized in
three-dimensional (3D) space, and lineage-specific transcriptional programs that
direct stem cell fate are implemented in this native 3D context. Here, we review
principles of 3D genome organization in mammalian cells. We focus on the
emerging relationship between genome organization and lineage-specific
transcriptional regulation, which we argue are inextricably linked. The relation between alterations in chromatin structure and changes in gene
expression during cell differentiation has served as a paradigm to understand
the link between genome organization and function. Yet, the factors involved and
the mechanisms by which the 3D organization of the nucleus is established remain
poorly understood. The use of Chromosome Conformation-Capture (3C)-based
approaches has resulted in a new appreciation of the role of architectural
proteins in the establishment of 3D genome organization. Architectural proteins
orchestrate higher-order chromatin organization through the establishment of
interactions between regulatory elements across multiple spatial scales. The
regulation of these proteins, their interaction with DNA, and their
co-occurrence in the genome, may be responsible for the plasticity of 3D
chromatin architecture that dictates cell and time-specific blueprints of gene
expression. Author information:
(1)Institute of Veterinary Biochemistry and Molecular Biology, University of
Zurich, 8057 Zurich, Switzerland; Molecular Life Science Program, Life Science
Zurich Graduate School, University of Zurich, 8057 Zurich, Switzerland.
(2)Institute of Veterinary Biochemistry and Molecular Biology, University of
Zurich, 8057 Zurich, Switzerland.
(3)Molecular Life Science Program, Life Science Zurich Graduate School,
University of Zurich, 8057 Zurich, Switzerland; Institute of Laboratory Animal
Science, University of Zurich, 8057 Zurich, Switzerland.
(4)Center for Microscopy and Image Analysis, University of Zurich, 8057 Zurich,
Switzerland.
(5)Department of Oncology, University Hospital Zurich, 8952 Schlieren,
Switzerland.
(6)Institute of Laboratory Animal Science, University of Zurich, 8057 Zurich,
Switzerland; Center for Applied Biotechnology and Molecular Medicine, University
of Zurich, 8057 Zurich, Switzerland; Division of Trauma Surgery, Center for
Clinical Research, University Hospital Zurich, 8091 Zurich, Switzerland.
(7)Institute of Veterinary Biochemistry and Molecular Biology, University of
Zurich, 8057 Zurich, Switzerland; Center for Applied Biotechnology and Molecular
Medicine, University of Zurich, 8057 Zurich, Switzerland. Electronic address:
[email protected]. Author information:
(1)1] Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla,
California 92093-0653, USA [2] Medical Scientist Training Program, University of
California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
(2)Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla, California
92093-0653, USA.
(3)1] Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla,
California 92093-0653, USA [2] Bioinformatics and Systems Biology Graduate
Program, University of California, San Diego, 9500 Gilman Drive, La Jolla,
California 92093, USA.
(4)The Morgridge Institute for Research, 309 North Orchard Street, Madison,
Wisconsin 53715, USA.
(5)Tsinghua University-Peking University Center for Life Sciences, School of
Life Sciences, Tsinghua University, Beijing 100084, China.
(6)Department of Chemical and Biomolecular Engineering, University of Illinois
at Urbana-Champaign, Urbana, Illinois 61801, USA.
(7)Laboratory of Immunogenetics, National Institute of Allergy and Infectious
Diseases, Twinbrook I NIAID Facility, Room 1417, 5640 Fishers Lane, Rockville,
Maryland 20852, USA.
(8)Howard Hughes Medical Institute, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, California 92037, USA.
(9)1] The Morgridge Institute for Research, 309 North Orchard Street, Madison,
Wisconsin 53715, USA [2] Department of Cell and Regenerative Biology, University
of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA
[3] Department of Molecular, Cellular, and Developmental Biology, University of
California Santa Barbara, Santa Barbara, California 93106, USA.
(10)1] Ludwig Institute for Cancer Research, 9500 Gilman Drive, La Jolla,
California 92093-0653, USA [2] University of California, San Diego School of
Medicine, Department of Cellular and Molecular Medicine, Institute of Genomic
Medicine, 9500 Gilman Drive, La Jolla, California 92093-0653, USA. |
What is the main biological function of the CRISPR-CAS9 genome editing system? | The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. The CRISPR/Cas9 system has attracted significant attention for its potential to transform genome engineering. It has been shown that the RNA-guided Cas9 nuclease can be employed to engineer the Drosophila genome, and that these modifications are efficiently transmitted through the germline. The CRISPR/Cas9 system has been reported to efficiently induce targeted gene disruption and homologous recombination in both prokaryotic and eukaryotic cells. The CRISPR/Cas9 system has been used to create knock-out alleles with great efficiency, and it has also been employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies. | Mice carrying mutations in multiple genes are traditionally generated by
sequential recombination in embryonic stem cells and/or time-consuming
intercrossing of mice with a single mutation. The CRISPR/Cas system has been
adapted as an efficient gene-targeting technology with the potential for
multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing
allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8
alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of
Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes
generated mice with biallelic mutations in both genes with an efficiency of 80%.
Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos
generated precise point mutations simultaneously in two target genes. Thus, the
CRISPR/Cas system allows the one-step generation of animals carrying mutations
in multiple genes, an approach that will greatly accelerate the in vivo study of
functionally redundant genes and of epistatic gene interactions. A simple and robust method for targeted mutagenesis in zebrafish has long been
sought. Previous methods generate monoallelic mutations in the germ line of F0
animals, usually delaying homozygosity for the mutation to the F2 generation.
Generation of robust biallelic mutations in the F0 would allow for phenotypic
analysis directly in injected animals. Recently the type II prokaryotic
clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a
targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system
in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein
that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four
endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached
75-99%, indicating that most cells contained biallelic mutations. Recessive
null-like phenotypes were observed in four of the five targeting cases,
supporting high rates of biallelic gene disruption. We also observed efficient
germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci
can be targeted simultaneously, resulting in multiple loss-of-function
phenotypes in the same injected fish. This CRISPR/Cas9 system represents a
highly effective and scalable gene knockout method in zebrafish and has the
potential for applications in other model organisms. The type II CRISPR/Cas9 system (clustered regularly interspaced short
palindromic repeats/CRISPR-associated) has recently emerged as an efficient and
simple tool for site-specific engineering of eukaryotic genomes. To improve its
applications in Drosophila genome engineering, we simplified the standard
two-component CRISPR/Cas9 system by generating a stable transgenic fly line
expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting
vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly
embryos, mutations were generated from site-specific DNA cleavages and
efficiently transmitted into progenies. Because Cas9 endonuclease is the
universal component of the type II CRISPR/Cas9 system, site-specific genomic
engineering based on this improved platform can be achieved with lower
complexity and toxicity, greater consistency, and excellent versatility. Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly
expanded the genome editing possibilities in model organisms such as zebrafish.
Both systems have recently been used to create knock-out alleles with great
efficiency, and TALENs have also been successfully employed in knock-in of DNA
cassettes at defined loci via homologous recombination (HR). Here we report
CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a
very high rate by homology-independent double-strand break (DSB) repair
pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA)
and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the
selected chromosomal integration site resulted in efficient targeted integration
of donor DNA. We successfully employed this approach to convert eGFP into Gal4
transgenic lines, and the same plasmids and sgRNAs can be applied in any species
where eGFP lines were generated as part of enhancer and gene trap screens. In
addition, we show the possibility of easily targeting DNA integration at
endogenous loci, thus greatly facilitating the creation of reporter and
loss-of-function alleles. Due to its simplicity, flexibility, and very high
efficiency, our method greatly expands the repertoire for genome editing in
zebrafish and can be readily adapted to many other organisms. Cas9/CRISPR has been reported to efficiently induce targeted gene disruption and
homologous recombination in both prokaryotic and eukaryotic cells. Thus, we
developed a Guide RNA Sequence Design Platform for the Cas9/CRISPR silencing
system for model organisms. The platform is easy to use for gRNA design with
input query sequences. It finds potential targets by PAM and ranks them
according to factors including uniqueness, SNP, RNA secondary structure, and AT
content. The platform allows users to upload and share their experimental
results. In addition, most guide RNA sequences from published papers have been
put into our database. Gene modifications in animal models have been greatly facilitated through the
application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type
II genome editing system has recently been applied in cell lines and
vertebrates. However, we still have very limited information about the
efficiency of mutagenesis, germline transmission rates and off-target effects in
genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in
zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The
efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in
of a protein tag provided by a donor oligonucleotide with knock-in efficiencies
of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%,
demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency
of the CRISPR/Cas9 system with limited off-target effects make it a powerful
genome engineering tool for in vivo studies. We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate
targeted genetic mutations in more than 85% of alleles. By targeting a
constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to
82% of cells, thereby allowing the study of genetic knockouts in a Drosophila
cell line for the first time. We have shown that homologous gene targeting is
possible at 1-4% efficiency using this system, allowing for the construction of
defined insertions and deletions. We demonstrate that a 1 kb homology arm
length is optimal for integration by homologous gene targeting, and demonstrate
its efficacy by tagging the endogenous AGO1 protein. This technology enables
controlled genetic manipulation in Drosophila cell lines, and its simplicity
offers the opportunity to study cellular phenotypes genome-wide. |
Does dronedarone affect T3 and T4 levels? | NO. | The present study investigated the effects of dronedarone and amiodarone on
plasma thyroid hormones and the possible consequences on the response of the
heart to ischemia. Amiodarone (30 mg/kg/day per os) or dronedarone (30 mg/kg/day
per os) were administered for 2 weeks in normal and thyroxine-treated animals
(25 microg/100 g body weight od sc, for 2 weeks), while animals without
amiodarone and dronedarone served as controls. Isolated rat hearts were perfused
in a Langendorff mode and subjected to 20 and 30 min of zero-flow global
ischemia followed by 45 min of reperfusion. Functional changes were assessed by
measuring left ventricular developed pressure (LVDP) under resting conditions
and in response to ischemia-reperfusion, LVDP%, as well as the severity of
ischemic contracture. Amiodarone resulted in increased T4, T4/T3 and rT3,
whereas dronedarone did not alter the thyroid hormone profile in normal animals.
In thyroxine-treated animals, amiodarone increased T4/T3 ratio but T4, T3 and
rT3 levels were not altered. Basal functional parameters and ischemic
contracture did not change by amiodarone and/or dronedarone neither in normal
nor in thyroxine-treated hearts. In normal hearts, postischemic functional
recovery, LVDP%, was not altered by amiodarone or dronedarone administration.
LVDP% was statistically higher in thyroxine-treated hearts than in normal and
this beneficial effect was not abolished by amiodarone or dronedarone treatment. The effects of dronedarone, a non-iodinated derivative of amiodarone, on
ventricular tachycardia and ventricular fibrillation post-myocardial infarction
are not well established. Fifty-five Wistar rats were randomly allocated to a
2-week oral treatment with either vehicle (n=18), amiodarone (30 mg/kg, n=20),
or dronedarone (30 mg/kg, n=17). After acute coronary artery ligation, a
single-lead electrocardiogram was continuously recorded for 24 h and episodes of
ventricular tachycardia/fibrillation as well as mortality rates were analysed.
Monophasic action potential recordings were obtained from the left ventricular
epicardium at baseline and 24 h post-myocardial infarction. Thyroid hormones and
catecholamines were measured using radioimmunoassay. Thyroid function was
similar in the 3 groups. Compared to controls, amiodarone and dronedarone
equally decreased the number of ventricular tachycardia/fibrillation episodes by
approximately 75%. Both agents prevented the increase in monophasic action
potential duration and in beat-to-beat variation. Norepinephrine levels were
lower only after amiodarone treatment. Despite the observed antiarrhythmic
effect, total mortality did not differ between groups (38.8% in controls, 30.0%
in the amiodarone group and 58.8% in the dronedarone group), because of excess
bradyarrhythmic mortality in both drug groups that reached significance in the
dronedarone group. Dronedarone and amiodarone display similar antiarrhythmic
efficacy post-myocardial infarction, partly by preventing repolarization
inhomogeneity. However, dronedarone increases bradyarrhythmic mortality possibly
secondary to its negative inotropic effects. |
What is 2d 4d ratio in athletes. | Lower 2D:4D ratio was reported to be lower in handball players, kabaddi players, varsity athletes, football players, soccer players and rugby players. Low 2D:4D ratio correlates with better performance and with enhanced sporting prowess, particularly with regard to activities requiring endurance and dependent upon slow-twitch muscles. | Contests in the animal world to determine social status almost exclusively
involve males, which points out that androgens may be indispensable in the
development of competitive instincts. In animal studies, it has been shown that
prenatal exposure to androgens may produce permanent changes toward more
aggressive behavior in adulthood. Thus, there is a strong suspicion that women
involved in competitive activities, such as sports, may have been exposed to
high androgen levels in utero. There is strong evidence that the ratio between
the second to fourth digits ratio (2D:4D ratio) correlates negatively with
intrauterine androgen concentrations and could potentially be used as a marker
for prenatal androgen exposure. Therefore, the purpose of our study was to test
the hypothesis that women engaged in sports have lower 2D:4D ratio-a marker of
high prenatal androgen exposure. We measured the 2D:4D ratios in elite and
non-elite female athletes and compared them with female individuals not engaged
in any sport activities. Our results showed that elite female athletes have
significantly lower left hand 2D:4D ratios compared to the control group (P <
0.05). Therefore, we can speculate that low 2D:4D ratio may be a positive
correlate of sports potential in females. Research particularly focusing on male athletes and popular sports (running and
soccer) suggests associations of lower (masculinized) second-to-fourth digit
ratio (2D:4D), a putative marker of prenatal androgen action, with better sports
performance. Studies focusing on women, non-mainstream sports, or controlling
for covariates relevant for sporting success are still sparse. This study
examined associations between 2D:4D and performance of both male and female
athletes active in fencing (a non-mainstream sport dominated by male
participants), while controlling for covariates. National fencing rankings and
2D:4D of 58 male and 41 female Austrian tournament fencers (mean age 24 years)
were correlated. Among female, but not male, fencers, lower 2D:4D was related to
better national fencing rankings. 2D:4D still accounted for incremental variance
(12%) in fencing success, when the effects of salient performance factors (age,
body mass index, years of fencing, training intensity, and the personality
variables achievement, control, harm avoidance, and social potency) were
controlled for (totaling 35% attributable variance). Athletes active in the most
aggressive form (the sabre) had lower 2D:4D than those active in the other forms
(épée and foil fencing). Sporting success in adult life might be partly
prenatally programmed via long-lasting extragenital effects of testosterone. Second-to-fourth digit ratio (2D:4D), a widely studied putative marker for
masculinization through prenatal androgen exposure, is lower (more masculinized)
in athletes than in general population controls, and athletes with lower 2D:4D
have higher sporting success. Occupations differ markedly in perceived
masculinity and actual maleness (sex ratios), but these givens have not yet been
picked up and utilized in 2D:4D research. Accordingly, this study extended
existing accounts on 2D:4D in athletes to a novel approach: 2D:4D and possible
relationships to a variety of candidate variables (demographic,
fertility-related, psychological, and other) were investigated in firefighters,
a highly male-dominated occupation. Contrary to expectation, 2D:4D in
firefighters (N = 134) was not lower than in local male population controls.
Lower 2D:4D corresponded to lower service ranks. Replicating previous findings
either unequivocally or partly, lower 2D:4D was associated with larger family
size, later sibling position, left-handedness, and higher scores in the
disinhibition component of sensation seeking. Not replicating prior evidence,
2D:4D was unrelated to body-mass index, offspring sex ratio, and sporting
performance level. Novel findings included low 2D:4D in those with low
relationship satisfaction and in cigarette smokers, especially among heavy
smokers. Absolute finger length, a positive correlate of pubertal-adolescent
androgen levels, was also considered. This marker showed negative associations
with relationship consensus and satisfaction and positive ones with perceived
quality of relationship alternatives and the experience seeking component of
sensation seeking. The merits of this additional marker, relative to 2D:4D, for
supplementing studies of possible sex-hormonal effects on personality and
directions for future inquiry along these lines are discussed. Research suggests that prenatal levels of testosterone are related to finger
length development and traits beneficial to athletic skill, such as power,
endurance, visual-spatial skills, or sensation seeking and domice behavior.
In men, the second digit to fourth digit ratio (2D:4D) has been shown to
correlate with success in competitive levels of football (soccer), which
suggests that the 2D:4D ratio is a possible marker for level of attainment in
sport. The purpose of this study was to explore the 2D:4D relationships between
sports and make comparisons with nonathletes. A multiple group posttest-only
design was used. Participants included 138 male volunteers with 92
intercollegiate National Collegiate Athletic Association division I athletes and
46 nonathletes who were not varsity athletes. The independent variable was group
(crew, football, gymnastics, soccer, nonathlete). The dependent variable was the
2D:4D ratio. No significant differences were noted between the athletes and
nonathletes (p = 0.182). Significant differences were found among the different
groups (p = 0.000), with significantly lower ratios between football and crew (p
= 0.000), football and nonathletes (p = 0.030), and gymnastics and crew (p =
0.001). This research provides a stronger level of evidence that the 2D:4D ratio
may help indicate potential athleticism or competition-level achievement, but
the external validity may be limited to only specific sports. The relative length of the second and fourth digits (2D:4D) is a putative marker
for prenatal testosterone. Low 2D:4D has been reported to correlate with high
performance in sport in general. Here, for the first time, we examine the
relationship between 2D:4D and performance in elite rugby players. The 44
players (28 forwards, 16 backs) were drawn from the Ospreys Rugby Union Club and
44 age-matched controls. The measures of performance comprised age-adjusted
number of international performances (caps) for Wales, a comparison of coaches'
first-choice League team with others, and the number of tries scored by backs in
club matches. Compared with controls, players were larger and had lower 2D:4D
for the right and left hand. With regard to number of caps, players with low
2D:4D in their right hand and low right 2D:4D compared with their left
(right - left 2D:4D difference) had high numbers of caps. First-choice players
did not differ significantly from second-choice players in their 2D:4D but they
did have a lower right - left 2D:4D difference than second-choice players. Low
right 2D:4D and low right - left 2D:4D difference were significantly linked with
large numbers of tries. We conclude that low right 2D:4D and low right - left
2D:4D difference are predictors of high rugby performance. Our objective was to propose a testable hypothesis arising from the recent
finding of a low index-to-ring finger ratio (2D:4D ratio) in ALS. The 2D:4D
ratio finding suggests that prenatal testosterone exposure may play a role in
the development of the disease. Research from other fields is presented to
suggest that healthy individuals with low 2D:4D ratio have enhanced sporting
prowess, particularly with regard to activities requiring endurance and
dependent upon slow-twitch muscles. Although studies are of varying quality,
some epidemiological findings in ALS also suggest enhanced sporting prowess, as
well as a higher risk of developing the disease among members of certain
physically active professions. If the 2D:4D finding survives replication then
this might explain the reported elevated risk of ALS among professional
athletes, the military, and manual professions. Such a relationship might also
explain why ALS patients were more likely to have been elite sportspeople in
younger life. This hypothesis may serve as a starting point for debate and
discussion over the nature of ALS risk factors, as well as generating a number
of specific testable hypotheses that may yield insight into the genesis of the
disease. BACKGROUND: Ratio of second and fourth digit (2D:4D) is known to be germane in
analyzing utero concentrations of testosterone and estrogen in human and other
vertebrates. 2D:4D had been linked to several traits like athletes' abilities,
reproductive success, risk of cancer and cardiovascular disease (CVD). Metabolic
syndrome (MetS) is a clustering of several cardiovascular risk factors. Waist
circumference (WC), neck circumference (NC), body mass index (BMI) and
waist-to-height ratio (WHtR) are important in measuring MetS. This study
investigated sexual dimorphism in 2D:4D and its relationship with MetS indices
and CVD factors among adult residing in Ilorin, North central Nigeria.
MATERIALS AND METHODS: This is a cross-sectional, stratified multi-staged
sampling study. Participants residing in different neighborhoods were visited at
home where finger lengths and anthropometric traits were measured. Participants
include 801 healthy adults aged 18-44 years (56% male) who had been living in
the area for more than 3 years.
RESULTS: Males showed significantly lower 2D:4D than females (unpaired t-test; t
[699] = 11.49, P = 0.001). A significant positive correlation was observed in
MetS markers and 2D:4D. WHtR showed the highest correlation with 2D:4D in male
(r = 0.461, P ≤ 0.001) and female (r = 0.408, P ≤ 0.001) when compared with BMI,
NC and WC. All positive correlations recorded in this study were high in male
and right hand.
CONCLUSION: Our results showed that 2D:4D is sexual dimorphic and right hand
2D:4D as a predictor of MetS is better. We concluded that 2D:4D is a proxy for
MetS and CVD risk factors in Ilorin. |
What is the association between moon cycle and rupture risk of intracranial aneurysms? | The lunar cycle seems to affect the incidence of intracranial aneurysm rupture, with the new moon being associated with an increased risk of aneurysmal SAH. | OBJECTIVE: To analyze the impact of the lunar cycle and season on the incidence
of aneurysmal subarachnoid hemorrhage (SAH).
PATIENTS AND METHODS: The medical records of 111 patients who were admitted over
a 5-year period to our department because of aneurysmal SAH were retrospectively
reviewed. The date of aneurysm rupture was matched with the corresponding season
and moon phase.
RESULTS: An incidence peak for aneurysm rupture (28 patients) was seen during
the phase of new moon, which was statistically significant (p < 0.001). In
contrast, no seasonal variation in the incidence of SAH was observed.
CONCLUSION: The lunar cycle seems to affect the incidence of intracranial
aneurysm rupture, with the new moon being associated with an increased risk of
aneurysmal SAH. |
What is the role of brain natriuretic peptide in traumatic brain injury patients ? | Brain natriuretic peptide concentrations are elevated in patients with traumatic brain during the acute phase and correlate with poor outcomes. In traumatic brain injury patients higher brain natriuretic peptide concentrations are associated with more extensive SAH, elevated ICP and hyponatremia. Brain natriuretic peptide may play an adaptive role in recovery through augmentation of cerebral blood flow. | BACKGROUND: Brain natriuretic peptide (BNP) is a potent natriuretic and
vasodilator factor which, by its systemic effects, can decrease cerebral blood
flow (CBF). In aneurysmal subarchnoid hemorrhage (aSAH), BNP plasma
concentrations were found to be associated with hyponatremia and were
progressively elevated in patients who eventually developed delayed ischemic
deficit secondary to vasospasm. The purpose of the present study was to evaluate
trends in BNP plasma concentrations during the acute phase following severe
(traumatic brain injury) TBI.
METHODS: BNP plasma concentration was evaluated in 30 patients with severe
isolated head injury (GCS<8 on admission) in four time periods after the injury
(period 1: days 1-2; period 2: days 4-5; period 3: days 7-8; period 4: days
10-11). All patients were monitored for ICP during the first week after the
injury.
FINDINGS: The initial BNP plasma concentrations (42+/-36.9 pg/ml) were 7.3 fold
(p<0.01) higher in TBI patients as compared to the control group (5.78+/-1.90
pg/ml). BNP plasma concentrations were progressively elevated through days 7-8
after the injury in patients with diffused SAH as compared to patients with mild
or no SAH (p<0.001) and in patients with elevated ICP as compared to patients
without elevated ICP (p<0.001). Furthermore, trends in BNP plasma concentrations
were significantly and positively associated with poor outcome.
INTERPRETATION: BNP plasma concentrations are elevated shortly after head injury
and are continuously elevated during the acute phase in patients with more
extensive SAH and in those with elevated ICP, and correlate with poor outcomes.
Further studies should be undertaken to evaluate the role of BNP in TBI
pathophysiology. There is emerging evidence to suggest that brain natriuretic peptide (BNP) is
elevated after acute brain injury, and that it may play an adaptive role in
recovery through augmentation of cerebral blood flow (CBF). Through a series of
experiments, we tested the hypothesis that the administration of BNP after
different acute mechanisms of central nervous system (CNS) injury could improve
functional recovery by improving CBF. C57 wild-type mice were exposed to either
pneumatic-induced closed traumatic brain injury (TBI) or collagenase-induced
intracerebral hemorrhage (ICH). After injury, either nesiritide (hBNP) (8
microg/kg) or normal saline were administered via tail vein injection at 30 min
and 4 h. The mice then underwent functional neurological testing via rotorod
latency over the following 5 days and neurocognitive testing via Morris water
maze testing on days 24-28. Cerebral blood flow (CBF) was assessed by laser
Doppler from 25 to 90 min after injury. After ICH, mRNA polymerase chain
reaction (PCR) and histochemical staining were performed during the acute injury
phase (<24 h) to determine the effects on inflammation. Following TBI and ICH,
administration of hBNP was associated with improved functional performance as
assessed by rotorod and Morris water maze latencies (p < 0.01). CBF was
increased (p < 0.05), and inflammatory markers (TNF-alpha and IL-6; p < 0.05),
activated microglial (F4/80; p < 0.05), and neuronal degeneration (Fluoro-Jade
B; p < 0.05) were reduced in mice receiving hBNP. hBNP improves neurological
function in murine models of TBI and ICH, and was associated with enhanced CBF
and downregulation of neuroinflammatory responses. hBNP may represent a novel
therapeutic strategy after acute CNS injury. BACKGROUND: According to the literature, serum beta-natriuretic peptide (BNP)
levels have been shown to increase in adult trauma patients, specifically for
those with traumatic brain injury and in those with intracranial hemorrhage. It
has been suggested that BNP levels may be an ideal serum marker for traumatic
brain injury. It may save time and radiation if the levels correlated with head
computed tomography (CT) scan findings, especially for pediatric patients who
have higher radiation risks. We hypothesized that serum BNP levels would be
elevated in patients with intracranial bleeding on head CT.
METHOD: Serum BNP levels were drawn from 95 consecutive "Level I status"
pediatric trauma patients immediately on presentation to the emergency
department. These patients had high impact mechanisms, were altered, or were
physiologically unstable. The findings of head CTs were recorded. Patients were
subsequently divided into a negative bleed or positive bleed group. Clinical
data such as Glasgow Coma Scale, loss of consciousness, and hospital course were
collected. Results were compared using Wilcoxon rank sum test and Spearman
correlation coefficients.
RESULTS: BNP levels did not increase significantly in the positive bleed group
(n = 21) compared with the negative bleed group (n = 74) (p = 0.48). BNP levels
did not correlate with loss of consciousness, Glasgow Coma Scale, Injury
Severity Score, or hospital stay.
CONCLUSION: BNP levels drawn at the time of the emergency department visit do
not seem to be a predictor for intracranial hemorrhage in pediatric trauma
patients. A head CT still remains the best diagnostic study for diagnosing
intracranial hemorrhage. BACKGROUND: The role of brain natriuretic peptide (BNP) after traumatic brain
injury (TBI) remains unclear, and its relationship with hyponatremia is still
controversial. The aim of this study is to investigate the secretion pattern of
N-terminal (NT)-proBNP in patients with TBI and to assess the relationship
between NT-proBNP, sodium balance, and intracranial pressure (ICP).
METHODS: We measured serum NT-proBNP levels of 84 patients with isolated TBI on
a daily basis from day 1 to day 14 after injury.
RESULTS: In average, the peak of BNP level was measured at 703.9 pg/mL±179.1
pg/mL on day 3 after injury, which was correlated to the severity of TBI. Among
patients with severe TBI, plasma NT-proBNP concentrations in patients with
hyponatremia were statistically higher than those without hyponatremia (p<0.05).
In the hyponatremic group, the plasma NT-proBNP increased to a peak of 1001.16
pg/mL±131.52 pg/mL within 48 hours after injury and maintained at a high level
for 3 days. In the normonatremic group, the plasma NT-proBNP reached a peak of
826.43 pg/mL±337.43 pg/mL on day 5 and quickly decreased thereafter. In
addition, we found plasma NT-proBNP concentrations in patients with ICP>15 mm Hg
were significantly higher than those in patients with ICP≤15 mm Hg (p<0.01).
CONCLUSIONS: This study provides evidence that BNP plasma concentrations
increase rapidly after TBI. Plasma BNP concentrations are correlated with
hyponatremia in severe TBI patients but not in mild and moderate TBI patients.
Furthermore, patients with elevated ICP have a higher serum BNP level in first 4
days after injury. |
Which molecule is targeted by Daratumumab? | Daratumumab, an investigated anti-cancer drug targeting CD38, has been of great interest in the treatment of CD38-expressing malignancies, especially multiple myeloma. | BACKGROUND: In our efforts to develop novel effective treatment regimens for
multiple myeloma we evaluated the potential benefits of combining the
immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel
human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via
antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity
and apoptosis.
DESIGN AND METHODS: To explore the effect of lenalidomide combined with
daratumumab, we first carried out standard antibody-dependent cell-mediated
cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+
multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from
patients were used as target cells. We also tested the effect of lenalidomide on
daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent
cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear
cells of multiple myeloma patients. Finally, we determined the
daratumumab-dependent cell-mediated cytotoxicity using peripheral blood
mononuclear cells of multiple myeloma patients receiving lenalidomide treatment.
RESULTS: Daratumumab-dependent cell-mediated cytotoxicity of purified primary
multiple myeloma cells, as well as of the UM-9 cell line, was significantly
augmented by lenalidomide pre-treatment of the effector cells derived from
peripheral blood mononuclear cells from healthy individuals. More importantly,
we demonstrated a clear synergy between lenalidomide and daratumumab-induced
antibody-dependent cell-mediated cytotoxicity directly in the bone marrow
mononuclear cells of multiple myeloma patients, indicating that lenalidomide can
also potentiate the daratumumab-dependent lysis of myeloma cells by activating
the autologous effector cells within the natural environment of maligt cells.
Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly
up-regulated in peripheral blood mononuclear cells derived from 3 multiple
myeloma patients during lenalidomide treatment.
CONCLUSIONS: Our results indicate that powerful and complementary effects may be
achieved by combining lenalidomide and daratumumab in the clinical management of
multiple myeloma. CD38, a type II transmembrane glycoprotein highly expressed in hematological
maligcies including multiple myeloma (MM), represents a promising target for
mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of
action of daratumumab, a novel, high-affinity, therapeutic human mAb against a
unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular
cytotoxicity in CD38-expressing lymphoma- and MM-derived cell lines as well as
in patient MM cells, both with autologous and allogeneic effector cells.
Daratumumab stood out from other CD38 mAbs in its strong ability to induce
complement-dependent cytotoxicity in patient MM cells. Importantly,
daratumumab-induced Ab-dependent cellular cytotoxicity and complement-dependent
cytotoxicity were not affected by the presence of bone marrow stromal cells,
indicating that daratumumab can effectively kill MM tumor cells in a
tumor-preserving bone marrow microenvironment. In vivo, daratumumab was highly
active and interrupted xenograft tumor growth at low dosing. Collectively, our
results show the versatility of daratumumab to effectively kill CD38-expressing
tumor cells, including patient MM cells, via diverse cytotoxic mechanisms. These
findings support clinical development of daratumumab for the treatment of
CD38-positive MM tumors. Multiple myeloma (MM) has been mostly incurable due to its highly complex and
heterogeneous molecular abnormalities and the support from myeloma
microenvironment factors. A therapeutic strategy which effectively targets
relevant and specific molecule to myeloma cells, and which is potent in
overcoming tumor microenvironment-mediated drug resistance needs to be
developed. One of the promising fields is the development of immunotherapy using
monoclonal antibodies (MoAbs) against myeloma-specific antigens. This review
focuses on the basic and clinical aspects of two emerging and promising novel
MoAbs for MM, elotuzumab which targets CS1 and daratumumab which targets CD38.
Both antigens are relatively specific to myeloma cells and expressed in more
than 90% of MM patients, and mediate adhesion of myeloma cells to bone marrow
stromal cells. We also discuss the unique characteristics of the two MoAbs by
comparing with other MoAbs being developed for MM. Multiple myeloma (MM) remains incurable despite important recent advances in
treatment due to its inherent resistance, characterized by highly complex and
heterogeneous molecular abnormalities, as well as the support from myeloma bone
marrow (BM) microenvironment. A novel therapeutic strategy that effectively
targets specific molecules on myeloma cells and also potentially overcomes tumor
microenvironment-mediated drug resistance and the downstream effects of genetic
instability is thus urgently needed. Over the last 2 years, an anti-CD38
monoclonal antibody daratumumab (DARA) has emerged as a breakthrough targeted
therapy for patients with MM. Early-stage clinical trials have found DARA to be
safe and to have encouraging clinical activity as a single agent and in
combination with lenalidomide in heavily pretreated, relapsed patients in whom
other novel agents (such as bortezomib, thalidomide and lenalidomide) as well as
stem cell transplant has already failed. DARA may, therefore, be the first mAb
with significant anti-MM activity both as a monotherapy and in combination. It
is currently being further evaluated both alone and in combination with
conventional and novel anti-MM agents as part of prospective clinical trials.
This review discusses the preclinical and clinical development of DARA, its
pathophysiological basis, and its prospects for future use in MM. The diagnosis and treatment of multiple myeloma (MM) are progressing
continuously. This article aims at summarizing the current status in the
diagnosis and treatment of MM, emphasizing a clinical point of view. Prognostic
factors can be determined by clinical parameters, molecular analyses and patient
characteristics (e.g. age and comorbidities). The international staging system
(ISS) and cytogenetics, such as the high-risk aberrations 17p deletion,
translocation (4;14) and insertion 1q21 > 2 copies, are key factors in risk
stratification of MM patients. Induction therapy based on novel agents, namely
bortezomib, followed by subsequent high-dose melphalan and autologous stem cell
transplantation is considered the standard of care for younger, newly diagnosed
MM patients (≤ 70 years). Transplant-ineligible patients should receive
thalidomide or bortezomib-based chemotherapy. The combination of bortezomib,
melphalan and prednisone (VMP) was shown to significantly improve overall
survival (OS) compared to melphalan and prednisone (MP, 56.4 vs. 43.1 months,
p = < 0.01). Recent results suggest that lenalidomide-based therapy not
incorporating alkylating agents might be a competitive alternative with a
favorable toxicity profile for transplant-ineligible patients. Maintece
therapies are of increasing clinical significance in MM as they have the ability
to prolong overall survival; however, thalidomide maintece therapy should not
be used in MM patients with high-risk cytogenetics as it shortens OS. Refractory
or relapsed MM treatment continues to improve with the development of second and
third generation immunomodulatory agents and proteasome inhibitors. For example,
pomalidomide and dexamethasone vs. high-dose dexamethasone significantly
improved OS (12.7 vs. 8.1 months, p = 0.03). Novel therapy strategies include
targeted and stroma-directed approaches. Antibodies targeting CS-1 (elotuzumab)
and CD38 (daratumumab) in particular are currently undergoing advanced clinical
phase II/III trials. Multiple myeloma (MM) remains mostly incurable despite the recent progress in
the treatment strategy. One of novel fields for anti-MM therapeutic strategy is
the development of immunotherapy using monoclonal antibodies (MoAbs) against
myeloma-specific antigens. This article focuses on the basic and clinical
aspects of several emerging and promising novel MoAbs for MM, such as elotuzumab
which targets CS1 and daratumumab which targets CD38. Both antigens are highly
expressed in more than 90% of MM patients, and the clinical trials have shown
promising anti-MM effects, especially in combination with immunomodulatory agent
lenalidomide. We also discuss the characteristics and the results of clinical
trials of other MoAbs, such as tabalumab against B cell activating factor or
dacetuzumab against CD40, being developed for MM. BACKGROUND: Daratumumab (DARA), a promising novel therapy for multiple myeloma,
is an IgG1κ monoclonal antibody that recognizes CD38 on myeloma cells. During
routine compatibility testing, we observed that the plasma of five of five
DARA-treated patients demonstrated a positive antibody screen and panreactivity
on red blood cell (RBC) panel testing. We hypothesized that the observed
panreactivity reflected DARA binding to CD38 on reagent RBCs, and we
investigated methods to prevent this binding.
STUDY DESIGN AND METHODS: DARA binding to CD38+ or CD38- HL60 cells was assessed
by flow cytometry. To remove cell surface CD38, cells were incubated with
dithiothreitol (DTT) or trypsin. Soluble CD38 or anti-DARA was used to
neutralize DARA in solution. Routine blood bank serologic methods were used to
test samples from DARA-treated patients and normal plasma samples spiked with
DARA and/or alloantibodies.
RESULTS: Normal plasma samples spiked with DARA (0.1-10 µg/mL) and incubated
with reagent RBCs recapitulated the interference observed with samples from
DARA-treated patients. Flow cytometry experiments confirmed DARA binding to
CD38+ HL60 cells, but not to CD38- controls. DTT treatment of CD38+ HL60 cells
reduced DARA binding by 92% by denaturing cell surface CD38. Treating
DARA-containing plasma with soluble CD38 or anti-DARA idiotype also inhibited
DARA binding.
CONCLUSION: DARA causes panreactivity in vitro by binding to CD38 on reagent
RBCs. Treating reagent RBCs with DTT is a robust method to negate the DARA
interference, enabling the safe provision of blood to DARA-treated patients.
Because DTT denatures Kell antigens, K- units are provided to these patients. No standard chemotherapy regimens have been defined yet for extranodal natural
killer/T cell lymphoma (ENKTL), and the prognosis of patients with advanced or
relapsed disease is very poor. Daratumumab, an investigated anti-cancer drug
targeting CD38, has been of great interest in the treatment of CD38-expressing
maligcies, especially multiple myeloma. In this study, we reviewed the
clinical data of 94 patients with ENKTL, investigated the expression of CD38,
and analyzed the prognostic value of CD38 expression. Forty-seven patients had
weak expression of CD38, and the other 47 patients had strong expression. The
complete response (CR) rate was significantly higher in patients who were
treated with asparaginase-based therapy (83.8 vs. 59.6 %, p = 0.025). There was
a trend towards higher CR rate in CD38 weak expression group (78.7 vs. 59.6 %,
p = 0.074). At a median follow-up time of 42 months, the 2-year and 5-year
progression-free survival (PFS) rates were 53.0 and 39.0 %, respectively, and
the 2-year and 5-year overall survival (OS) rates were 68.0 and 58.0 %,
respectively. In multivariate survival analysis including CD38 expression
status, International Prognostic Index (IPI) score, local tumor invasion, and
chemotherapy regimens, it was found that strong expression of CD38 and
non-asparaginase-based chemoregimens were independent adverse prognostic factors
for PFS (p = 0.009 and 0.027, respectively), while local tumor invasion and
higher IPI score were independent adverse prognostic factors for OS (p = 0.002
and 0.035, respectively). In subgroup analysis, strong expression of CD38
significantly correlated with inferior survival outcomes in patients without
local tumor invasion (p = 0.011) or with stage I-II disease (p = 0.008). In
conclusion, we firstly found that the majority of ENKTL cases were CD38
positive, with half had strong expression of CD38, which significantly
correlated with poor outcomes, indicating the potential role of CD38 as a
therapy target for ENKTL. Monoclonal antibodies (mAb) have had tremendous success in treating a variety of
cancers over the past twenty years. Yet despite their widespread clinical use,
which includes treatments for haematological maligcies, there are still no
approved mAb therapies for multiple myeloma (MM). This is likely to change
within the next few years with a number of mAb therapies being assessed in late
stage clinical trials, most notably, the anti-CS-1 mAb, elotuzumab, and the
anti-CD38 mAb, daratumumab, which are currently being evaluated in Phase III
clinical trials for MM. In this review, we will discuss the preclinical and
clinical development of MDX-1097, a Phase II candidate which targets cell
membrane-associated kappa immunoglobulin free light chains expressed on the
surface of MM cells. BACKGROUND: Monoclonal antibodies (MoAbs) are increasingly integrated in the
standard of care. The notion that therapeutic MoAbs can interfere with clinical
laboratory tests is an emerging concern that requires immediate recognition and
the development of appropriate solutions. Here, we describe that treatment of
multiple myeloma patients with daratumumab, a novel anti-CD38 MoAb, resulted in
false-positive indirect antiglobulin tests (IATs) for all patients for 2 to 6
months after infusion. This precluded the correct identification of irregular
blood group antibodies for patients requiring blood transfusion.
STUDY DESIGN AND METHODS: The IAT was performed using three- and 11-donor-cell
panels. Interference of daratumumab and three other anti-CD38 MoAbs was studied
using fresh-frozen plasma spiked with different MoAb concentrations.
Additionally it was tested whether two potentially neutralizing agents,
anti-idiotype antibody and recombit soluble CD38 (sCD38) extracellular
domain, were able to inhibit the interference.
RESULTS: The CD38 MoAbs caused agglutination in the IAT in a dose-dependent
manner. Addition of an excess of anti-idiotype antibodies or sCD38 protein to
the test abrogated CD38 MoAb interference and successfully restored irregular
antibody screening and identification.
DISCUSSION: CD38 MoAb therapy causes false-positive results in the IAT. The
reliability of the test could be restored by adding a neutralizing agent against
the CD38 MoAb to the patient's plasma. This study emphasizes that during drug
development, targeted therapeutics should be investigated for potential
interference with laboratory tests. Clinical laboratories should be informed
when patients receive MoAb treatments and matched laboratory tests to prevent
interference should be employed. Despite the recent major advancement in therapy for multiple myeloma, it remains
an incurable disease. There remains an unmet need for novel therapies that
target different mechanisms of action. Immunotherapy with monoclonal antibodies
is a promising area of development and will expand our therapeutic armamentarium
in the fight against myeloma. Daratumumab is a novel, high-affinity, therapeutic
human monoclonal antibody against unique CD38 epitope with broad-spectrum
killing activity. It has a favorable safety profile as monotherapy in patients
with relapsed/refractory myeloma and also demonstrates significant single-agent
activity. Abundant preclinical data supports its use in combination therapy and
clinical studies on various exciting combinations are underway. This review
focuses on the CD38 antigen and its targeting with daratumumab and provides an
update on the results of recent clinical studies involving daratumumab. |
Which mutation is associated with PLMS (periodic limb movements in sleep)? | missense substitution, Met1Val (M1V), was identified in the DCX gene | Mutations of the DCX gene (Xp22.3) cause X-linked lissencephaly in males and
double cortex syndrome (DCS) or subcortical band heterotopia (SBH) in females.
SBH is characterized by bilateral bands of grey matter interposed in the white
matter between the cortex and the lateral ventricles. The main clinical
manifestation in patients with SBH is epilepsy, which may be partial or
generalized and is intractable in approximately 65% of the patients. An
association of periodic limb movements (PLMs) and SBH has not been documented
previously. We describe a 2-year-old girl affected by SBH with epilepsy and
periodic limb movements (PLMs), in whom a novel "de novo" missense substitution,
Met1Val (M1V), was identified in the DCX gene. Physiopathological links between
PLMs and SBH are discussed. |
Which DNA repair system is involved in HNPCC? | In HNPCC families, germline mutations in any of four genes encoding proteins of a specialized DNA repair system, the mismatch repair, predispose to cancer development. | Lynch syndrome, or hereditary nonpolyposis colon cancer (HNPCC), is an
autosomal-domit disease accounting for approximately 1-5% of all colorectal
cancer cases. Due to the lack of pathognomonic morphological or biomolecular
markers, HNPCC has traditionally posed unique problems to clinicians and
geneticists alike, both in terms of diagnosis and clinical management. Recently,
novel insight into the pathogenesis of this syndrome has been provided by the
identification of its molecular basis. In HNPCC families, germline mutations in
any of four genes encoding proteins of a specialized DNA repair system, the
mismatch repair, predispose to cancer development. Mutations in mismatch repair
genes lead to an overall increase of the mutation rate and are associated with a
phenotype of length instability of microsatellite loci. The present report
summarizes the clinicopathological aspects of HNPCC and reviews the most recent
molecular and biochemical findings. Mutations of tumor suppressor genes, of the mismatch DNA repair system, and of
the TGF-beta-II-receptor are the main causes for a higher risk of colorectal
cancer. Among mutations of the Ape gene, which characterize the clinical
manifestation of the familial polyposis (FAP), point mutations are dominating
which create new stop codons or arise from deletions or insertions of
nucleotides causing frame shifts. Because the binding site of beta-catenin is
localized in the C-terminus of the Ape protein, disturbances result in the
cellular signal transfer from its loss. Consequently, the interactions of the
usually formed Ape-beta-catenin complex with the cytoskeleton and the cadherin
system in the plasma membrane as well as the translocation of beta-catenin into
the nucleus cannot be realized. Mutations in the genes of the mismatch DNA
repair system and of the TGF-beta-II-receptor, the main defects of the HNPCC
(hereditary nonpolyposis colorectal cancer), are exclusively identified in
sequences of microsatellites. Because the majority of Apc gene mutations is also
localized in repetitive motifs even in CpG islands primary disturbances are to
postulate in the methylation pattern of the genes producing germline and somatic
mutations. Generally, complexly connected reactions are involved in this cascade
of colorectal cancer genesis. This fact explains the relatively late clinical
manifestation of the disease and offers the possibility to identify carriers
with an increased risk of colorectal cancer development in order to integrate
them into a programme of control and preventive medicine. Beside the known
treatment by surgery and cytostatics, inhibitors of prostaglandin synthesis gain
therapeutic significance. Cancerogenesis can be efficiently suppressed by
inhibition of the COX-2-induction (cyclo-oxygenase-2). There is a lack of
clinical experience for a decision whether a high intraluminal level of butyrate
in the large intestine can delay colorectal carcinogenesis. |
Mention the only available genomics and developmental transcriptomics resource for the urochordate Oikopleura dioica | OikoBase (http://oikoarrays.biology.uiowa.edu/Oiko/) is a tiling array-based genome browser resource for Oikopleura dioica, a metazoan belonging to the urochordates, the closest extant group to vertebrates. OikoBase facilitates retrieval and mining of a variety of useful genomics information and will provide a valuable resource for research in chordate development, genome evolution and plasticity and the molecular ecology of this important marine planktonic organism. | |
What is the association between NT-proBNP and cognitive function? | Greater NT-proBNP serum concentration is associated with poorer cognitive function and cognitive decline. In community-dwelling older adults, greater NT-proBNP levels were strongly associated with poor cognitive function independently from age, sex, education, hypertension, body mass index, exercise, alcohol use, smoking, low density lipoprotein cholesterol, creatinine clearance, and previous cardiovascular disease. However, other authors did not find an association between NT-proBNP and severe cognitive impairment (SCI). | BACKGROUND: Serum N-terminal-pro brain natriuretic peptide (NT-proBNP) is
regarded as a marker of vascular disease and has previously been shown to
exhibit an increased frequency of pathological values in elderly patients with
mental illness with vascular disease compared to patients without vascular
disease. Vascular disease plays an important role in cognitive impairment in
elderly patients with mental illness.
METHOD: We have investigated the relation between NT-proBNP, vascular disease
and cognition in consecutively enrolled elderly patients with mental illness.
RESULTS: NT-proBNP level is increased in patients with vascular disease compared
to patients without vascular disease, and a logistic regression analysis showed
that NT-proBNP was a significant predictor of vascular disease. However,
NT-proBNP level did not predict cognition as assessed by MMSE score. NT-proBNP
level also showed a highly significant relation to mortality in all patients.
CONCLUSION: Determinations of NT-proBNP could be used in elderly patients with
mental illness to detect patients in need of control and treatment of vascular
risk factors. The levels of NT-proBNP may also provide prognostic information. BACKGROUND: Clinicopathological studies suggest that Alzheimer's disease (AD)
pathology begins ∼10-15 years before the resulting cognitive impairment draws
medical attention. Biomarkers that can detect AD pathology in its early stages
and predict dementia onset would, therefore, be invaluable for patient care and
efficient clinical trial design. We utilized a targeted proteomics approach to
discover novel cerebrospinal fluid (CSF) biomarkers that can augment the
diagnostic and prognostic accuracy of current leading CSF biomarkers (Aβ42, tau,
p-tau181).
METHODS AND FINDINGS: Using a multiplexed Luminex platform, 190 analytes were
measured in 333 CSF samples from cognitively normal (Clinical Dementia Rating
[CDR] 0), very mildly demented (CDR 0.5), and mildly demented (CDR 1)
individuals. Mean levels of 37 analytes (12 after Bonferroni correction) were
found to differ between CDR 0 and CDR>0 groups. Receiver-operating
characteristic curve analyses revealed that small combinations of a subset of
these markers (cystatin C, VEGF, TRAIL-R3, PAI-1, PP, NT-proBNP, MMP-10, MIF,
GRO-α, fibrinogen, FAS, eotaxin-3) enhanced the ability of the best-performing
established CSF biomarker, the tau/Aβ42 ratio, to discriminate CDR>0 from CDR 0
individuals. Multiple machine learning algorithms likewise showed that the novel
biomarker panels improved the diagnostic performance of the current leading
biomarkers. Importantly, most of the markers that best discriminated CDR 0 from
CDR>0 individuals in the more targeted ROC analyses were also identified as top
predictors in the machine learning models, reconfirming their potential as
biomarkers for early-stage AD. Cox proportional hazards models demonstrated that
an optimal panel of markers for predicting risk of developing cognitive
impairment (CDR 0 to CDR>0 conversion) consisted of calbindin, Aβ42, and age.
CONCLUSIONS/SIGNIFICANCE: Using a targeted proteomic screen, we identified novel
candidate biomarkers that complement the best current CSF biomarkers for
distinguishing very mildly/mildly demented from cognitively normal individuals.
Additionally, we identified a novel biomarker (calbindin) with significant
prognostic potential. BACKGROUND: Natriuretic peptides have prognostic value across a wide spectrum of
cardiovascular diseases and may predict cognitive dysfunction in patients with
cardiovascular disease, even in the absence of previous stroke. Little is known
about the association of natriuretic peptides with cognitive function in
community-dwelling adults. We assessed the association between N-terminal pro
B-type natriuretic peptide (NT-proBNP) levels and cognitive function in
community-dwelling ambulatory older adults in the Rancho Bernardo Study.
METHODS: We studied 950 men and women, aged 60 years and older, who attended a
research clinic visit where a medical history and examination were performed,
and blood for cardiovascular disease risk factors and NT-proBNP levels were
obtained. Three cognitive function tests were administered: the Mini Mental
State Examination (MMSE), Trail-Making Test B (Trails B), and Category Fluency.
RESULTS: Participants with high NT-proBNP levels (≥450 pg/mL; n=198) were older
and had a higher prevalence of coronary heart disease (12% vs 30%), and stroke
(5% vs 11%; P≤.001 for both). In unadjusted analyses, all 3 cognitive function
test scores were significantly associated with NT-proBNP levels (P<.001). After
adjusting for age, sex, education, hypertension, body mass index, exercise,
alcohol use, smoking, low density lipoprotein cholesterol, creatinine clearance,
and previous cardiovascular disease, elevated NT-proBNP levels remained
independently associated with poor cognitive performance on MMSE (odds ratio
[OR] 2.0; 95% confidence interval [CI], 1.1-3.6; P=.02) and Trails B (OR 1.7;
95% CI, 1.2-2.7; P=.01), but not Category Fluency (OR 1.4; 95% CI, 0.9-2.2;
P=.19). Results were unchanged after excluding the 6% of participants with a
history of stroke.
CONCLUSIONS: NT-proBNP levels were strongly and independently associated with
poor cognitive function in community-dwelling older adults. BACKGROUND: Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) is
regarded as a sensitive marker of cardiovascular disease. Vascular disease plays
an important role in cognitive impairment.
METHOD: In 447 elderly patients with mental illness, serum NT-proBNP level and
the presence or absence of vascular disease according to the medical record were
used to categorize patients in different subgroups of vascular disease.
RESULTS AND CONCLUSION: Patients with vascular disease and elevated serum
NT-proBNP level had a lower cognition level, shorter survival time, lower renal
function and a higher percentage of pathological brain imaging than patients
with vascular disease and normal NT-proBNP level. Thus, elevated serum NT-proBNP
level might be helpful to detect patients who have a more severe cardiovascular
disease. BACKGROUND: Type 2 diabetes mellitus is associated with risk of congestive heart
failure (CHF), cognitive dysfunction and depression. CHF itself is linked both
to poor cognition and depression. The ventricular N-terminal pro-brain
natriuretic peptide (NT-proBNP) is a marker of CHF, suggesting potential as a
marker for cognitive impairment and/or depression. This was tested in the
Edinburgh Type 2 Diabetes Study (ET2DS).
METHODOLOGY AND PRINCIPAL FINDINGS: Cross-sectional analysis of 1066 men and
women aged 60-75 with type 2 diabetes. Results from seven neuropsychological
tests were combined in a standardised general cognitive ability factor, 'g'. A
vocabulary-based test estimated pre-morbid cognitive ability. The Hospital
Anxiety and Depression Scale (HADS) assessed possible depression. After
adjustment for age and sex, raised plasma NT-proBNP was weakly associated with
lower 'g' and higher depression scores (ß -0.09, 95% CI -0.13 to -0.03,
p = 0.004 and ß 0.08, 95% CI 0.04 to 0.12, p<0.001, respectively). Comparing
extreme quintiles of NT-proBNP, subjects in the highest quintile were more
likely to have reduced cognitive ability (within the lowest tertile of 'g') and
'possible' depression (HADS depression ≥8) (OR 1.80; 95% CI: 1.20, 2.70;
p = 0.005 and OR 2.18; 95% CI: 1.28, 3.71; p = 0.004, respectively).
Associations persisted when pre-morbid ability was adjusted for, but as expected
were no longer statistically significant following the adjustment for
diabetes-related and vascular co-variates (β -0.02, 95% CI -0.07 to 0.03, p>0.05
for 'g'; β 0.03, 95% CI -0.02 to 0.07, p>0.05 for depression scores).
CONCLUSION: Raised plasma NT-proBNP was weakly but statistically significantly
associated with poorer cognitive function and depression. The prospective phases
of the ET2DS will help determine whether or not NT-proBNP can be considered a
risk marker for subsequent cognitive impairment and incident depression and
whether it provides additional information over and above traditional risk
factors for these conditions. AIMS: Up to 50% of patients with heart failure (HF) may suffer from severe
cognitive impairment (SCI), but longitudinal studies are sparse, and effects of
changes in HF severity on cognitive function are unknown. Therefore, we assessed
the prevalence of SCI in HF patients, its relationship with HF severity, its
effects on morbidity and mortality, and the relationship between changes in HF
severity and cognitive function.
METHODS AND RESULTS: We included 611 patients from the Trial of Intensified
versus standard Medical therapy in Elderly patients with Congestive Heart
Failure (TIME-CHF) and assessed cognitive function [Hodkinson Abbreviated Mental
Test (AMT)] in relation to severity of HF (NYHA class, NT-proBNP) at baseline
and 18 months (n = 382) and effects on hospitalization-free survival and
mortality. SCI (i.e. AMT score ≤ 7) was present in 9.2% of patients at baseline,
but only 20% of them had a diagnosis of dementia. Prevalence of SCI remained
stable during follow-up. SCI was present at baseline more often in NYHA IV
patients compared with NYHA II [odds ratio 2.94; 95% confidence interval (CI)
1.15-7.51, P = 0.025], but it was not related to NT-proBNP levels. SCI was
related to higher mortality (hazard ratio 1.53, 95% CI 1.02-2.30, P = 0.04), but
not hospitalization-free survival. Changes in HF severity were not significantly
related to changes in cognitive function.
CONCLUSION: SCI is a frequent, but often unrecognized finding in HF patients,
but the influence of HF severity and its changes on cognitive function were less
than hypothesized. Trial registration ISRCTN43596477. Alzheimer's disease (AD) is a severe neurodegenerative disease. Cerebrovascular
changes often accompany AD-related pathology. Despite a considerable progress in
the diagnostic accuracy of AD, no blood biomarkers have been established so far.
The aim of the present study was to search for changes in plasma levels of 27
vascular-related proteins of healthy controls, patients with mild cognitive
impairment (MCI) and AD. In a sample of 80 participants we showed that out of
these 27 proteins, six proteins were slightly changed (up to 1.5×) in AD
(alpha2-macroglobulin, apolipoprotein-A1, plasminogen activator inhibitor, RAGE,
Tissue Inhibitors of Metalloproteinases-1 and Trombospondin-2) and one marker
(serum amyloid A) was enhanced up to 6× but with a very high variance. However,
N-terminal pro-brain natriuretic peptide (NT-proBNP) was significantly enhanced
both in MCI and AD patients (1.9×). In a second analysis of a sample of 110
subjects including younger healthy controls, we confirmed that NT-proBNP has the
potential to be a stable candidate protein for both diagnosis and AD disease
progression. OBJECTIVE: Elevated levels of N-terminal pro-brain natriuretic peptide
(NT-proBNP) are associated with cognitive impairment, which might be explained
by cardiovascular diseases or risk factors. The aim of this study was to
investigate the association of NT-proBNP with cognitive function and decline in
older adults at high risk of cardiovascular disease.
METHODS: We studied 5,205 men and women (mean age = 75 years) who were recruited
into the PROspective Study of Pravastatin in the Elderly at Risk. All
participants had pre-existing cardiovascular disease or risk factors thereof.
Four domains of cognitive function were tested at baseline and repeated during a
follow-up period of 3.2 years.
RESULTS: Participants with higher NT-proBNP (≥450ng/l) had worse baseline
cognitive function, including reaction time (mean difference high vs low
group = 3.07 seconds, 95% confidence interval [CI] = 0.83 to 5.32), processing
speed (-1.02 digits coded, 95% CI = -1.65 to -0.39), and immediate memory (-0.13
pictures remembered, 95% CI = -0.29 to 0.04). There was no significant
difference in delayed memory (-0.14, 95% CI = -0.38 to 0.10) between the
NT-proBNP groups. Participants with higher NT-proBNP had a steeper cognitive
decline, including reaction time (mean annual change high vs low group = 0.60
seconds, 95% CI = 0.14 to 1.07), processing speed (-0.15 digits coded, 95%
CI = -0.25 to -0.05), immediate memory (-0.05 pictures remembered, 95%
CI = -0.09 to 0.00), and delayed memory (-0.05 pictures remembered, 95%
CI = -0.11 to 0.01). Associations were independent of cardiovascular diseases
and risks.
INTERPRETATION: Higher NT-proBNP associates with worse cognitive function and
steeper cognitive decline, independent of cardiovascular diseases and risks.
Further studies to unravel the underlying mechanisms are warranted. |
List functional roles of the FtsZ protein. | Four major roles of FtsZ have been characterized: cell elongation, GTPase, cell division, and bacterial cytoskeleton. | BACKGROUND: In Bacillus mycoides, as well as in other members of the B. cereus
group, the tubulin-like protein of the division septum FtsZ is encoded by the
distal gene of the cluster division and cell wall (dcw). Along the cluster the
genes coding for structural proteins of the division apparatus are intermingled
with those coding for enzymes of peptidoglycan biosynthesis, raising the
possibility that genes with this different function might be coexpressed.
Transcription of ftsZ in two model bacteria had been reported to differ: in B.
subtilis, the ftsZ gene was found transcribed as a bigenic mRNA in the AZ
operon; in E. coli, the transcripts of ftsZ were monogenic, expressed by
specific promoters. Here we analyzed the size and the initiation sites of RNAs
transcribed from ftsZ and from other cluster genes in two B. mycoides strains,
DX and SIN, characterized by colonies of different chirality and density, to
explore the correlation of the different morphotypes with transcription of the
dcw genes.
RESULTS: In both strains, during vegetative growth, the ftsZ-specific RNAs were
composed mainly of ftsZ, ftsA-ftsZ and ftsQ-ftsA-ftsZ transcripts. A low number
of RNA molecules included the sequences of the upstream murG and murB genes,
which are involved in peptidoglycan synthesis. No cotranscription was detected
between ftsZ and the downstream genes of the SpoIIG cluster. The monogenic ftsZ
RNA was found in both strains, with the main initiation site located inside the
ftsA coding sequence. To confirm the promoter property of the site, a B.
mycoides construct carrying the ftsA region in front of the shortened ftsZ gene
was inserted into the AmyE locus of B. subtilis 168. The promoter site in the
ftsA region was recognized in the heterologous cellular context and expressed as
in B. mycoides.
CONCLUSIONS: The DX and SIN strains of B. mycoides display very similar RNA
transcription specificity. The ftsZ messenger RNA can be found either as an
independent transcript or expressed together with ftsA and ftsQ and, in low
amounts, with genes that are specific to peptidoglycan biosynthesis. Bacterial cell division involves a complex and dynamic sequence of events
whereby polymers of the protein FtsZ assemble at the division plane and
rearrange to achieve the goal of contracting the cell membrane at the site of
cell division, thus dividing the parent cell into two daughter cells. We present
a mathematical model (which we refer to as CAM-FF: Critical Accumulation of
Membrane-bound FtsZ Fibres) of the assembly of the contractile ring in terms of
the accumulation of short linear polymers of FtsZ that associate and dissociate
from the cell membrane. In prokaryotes, the biochemical function of FtsZ is
thought to underpin the assembly and at least the initial kinetic force of ring
contraction. Our model extends earlier work of Surovtsev et al. [PLoS Comput.
Biol., 2008, 4, e1000102] by adding (i) the kinetics of FtsZ accumulation on
cell membrane anchor proteins and (ii) the physical forces required to deform
the cell against its surface tension. Moreover, we provide a more rigorous
treatment of intracellular diffusion and we revise some of the model parameter
values in light of the experimental evidence now available. We derive a critical
contraction parameter which links the chemical population dynamics of
membrane-bound FtsZ molecules to the force of contraction. Using this parameter
as a tool to predict the ability of the cell to initiate division, we are able
to predict the division outcome in cells depleted of key FtsZ-binding proteins. Chlamydiae are important pathogens and symbionts with unique cell biological
features. They lack the cell-division protein FtsZ, and the existence of
peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG
together function in orchestrating cell division and maintaining cell shape in
almost all other bacteria. Using electron cryotomography, mass spectrometry and
fluorescent labelling dyes, here we show that some environmental chlamydiae have
cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG
synthesis inhibitor) leads to lower infection rates and aberrant cell shapes,
suggesting that PG synthesis is crucial for the chlamydial life cycle. Our
findings demonstrate for the first time the presence of PG in a member of the
Chlamydiae. They also present a unique example of a bacterium with a PG sacculus
but without FtsZ, challenging the current hypothesis that it is the absence of a
cell wall that renders FtsZ non-essential. Bacterial cytokinesis depends upon the tubulin-like GTPase FtsZ, which
polymerizes into an annular structure at midcell (the Z-ring) that defines the
division site. The Z-ring nucleates assembly of downstream machinery required
for cell wall synthesis and membrane fission, but may also generate constrictive
force. Recent high-resolution imaging of FtsZ in vivo has begun to illuminate
the organization of filaments within the Z-ring. This in vivo work has been
complemented by reconstitution of Z-rings in vitro to demonstrate the
force-generating capacity of FtsZ and explore its mechanism of action. Despite
these technical advances, whether FtsZ-mediated force generation is required for
cytokinesis and how Z-ring structure and constriction are mechanistically linked
to cell wall remodeling are open questions. Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of
mild and severe diseases for which no vaccine is yet available. We recently
reported the identification of three protein antigens that in combination
conferred wide protection against GAS infection in mice. Here we focused our
attention on the characterization of one of these three antigens, Spy0269, a
highly conserved, surface-exposed, and immunogenic protein of unknown function.
Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial
chains, which is indicative of an impaired capacity of the knockout mutant to
properly divide. Confocal microscopy and immunoprecipitation experiments
demonstrated that the protein was mainly localized at the cell septum and could
interact in vitro with the cell division protein FtsZ, leading us to hypothesize
that Spy0269 is a member of the GAS divisome machinery. Predicted structural
domains and sequence homologies with known streptococcal adhesins suggested that
this antigen could also play a role in mediating GAS interaction with host
cells. This hypothesis was confirmed by showing that recombit Spy0269 could
bind to mammalian epithelial cells in vitro and that Lactococcus lactis
expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro
and to mice nasal mucosa in vivo. On the basis of these data, we believe that
Spy0269 is involved both in bacterial cell division and in adhesion to host
cells and we propose to rename this multifunctional moonlighting protein as
SpyAD (Streptococcus pyogenes Adhesion and Division protein). Cell division in bacteria is driven by a cytoskeletal ring structure, the Z
ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation
must be tightly regulated to ensure faithful cell division, and several
mechanisms that influence the positioning and timing of Z-ring assembly have
been described. Another important but as yet poorly understood aspect of cell
division regulation is the need to coordinate division with cell growth and
nutrient availability. In this study, we demonstrated for the first time that
cell division is intimately linked to central carbon metabolism in the model
Gram-positive bacterium Bacillus subtilis. We showed that a deletion of the gene
encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of
glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant
and has significant effects on Z-ring formation in wild-type B. subtilis cells.
Addition of exogenous pyruvate restores normal division in the absence of the
pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the
coordination of bacterial growth and division. Our results support a model in
which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually
metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown
that this protein localizes over the nucleoid in a pyruvate-dependent manner and
may stimulate more efficient Z-ring formation at the cell center under
nutrient-rich conditions, when cells must divide more frequently.
IMPORTANCE: How bacteria coordinate cell cycle processes with nutrient
availability and growth is a fundamental yet unresolved question in
microbiology. Recent breakthroughs have revealed that nutritional information
can be transmitted directly from metabolic pathways to the cell cycle machinery
and that this can serve as a mechanism for fine-tuning cell cycle processes in
response to changes in environmental conditions. Here we identified a novel link
between glycolysis and cell division in Bacillus subtilis. We showed that
pyruvate, the final product of glycolysis, plays an important role in
maintaining normal division. Nutrient-dependent changes in pyruvate levels
affect the function of the cell division protein FtsZ, most likely by modifying
the activity of an enzyme that metabolizes pyruvate, namely, pyruvate
dehydrogenase E1α. Ultimately this system may help to coordinate bacterial
division with nutritional conditions to ensure the survival of newborn cells. Antibiotic resistance to Mycobacterium tuberculosis is a growing problem.
Therefore, development of new anti-tuberculosis antibiotics is urgent for the
control of tuberculosis (TB) infections. FtsZ, the homolog of eukaryotic
tubulin, is a GTPase that assembles into cytokinetic Z rings essential for cell
division in prokaryotic cells. FtsZ (filamentous temperature-sensitive protein
Z) polymerizes in a GTP-dependent manner, and polymerization of FtsZ forms into
dynamic protofilaments. In this study, we screened 20,000 compounds to identify
inhibitors of GTPase activity of M. tuberculosis FtsZ. We found that 297F
inhibited GTPase and polymerization of FtsZ, and reduced the amount of FtsZ
polymers. Furthermore, 297F has anti-TB activity with low cytotoxicity and shows
no antibacterial activities toward other Gram-positive or Gram-negative strains.
In vitro, 297F also induced filamentation in Mycobacterium smegmatis. All
results suggest that 297F inhibits bacterial proliferation by targeting M.
tuberculosis FtsZ and it may be useful as a lead compound for developing anti-TB
agents. Membranes determine two-dimensional and three-dimensional biochemical reaction
spaces in living systems. Defining size and shape of surfaces and volumes
encompassed by membrane is of key importance for cellular metabolism and
homeostasis, and the maintece and controlled transformation of membrane
shapes are coordinated by a large number of different protein assemblies. The
orchestration of spatial elements over distances orders of magnitudes larger
than protein molecules, as required for cell division, is a particularly
challenging task, requiring large-scale ordered protein filaments and networks.
The structure and function of these networks, particularly of cytoskeletal
elements, have been characterized extensively in cells and reconstituted
systems. However, their co-reconstitution with membranes from the bottom-up
under defined conditions, to elucidate their mode of action in detail, is still
a relatively new field of research. In this short review, we discuss recent
approaches and achievements with regard to the study of cytoskeletal protein
assemblies on model membranes, with specific focus on contractile elements as
those based on the bacterial division FtsZ protein and eukaryotic actomyosin
structures. In Escherichia coli, initial assembly of the Z ring for cell division requires
FtsZ plus the essential Z ring-associated proteins FtsA and ZipA.
Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP
binding pocket and result in cell division arrest at non-permissive
temperatures. We found that purified wild-type FtsA bound and hydrolysed ATP,
whereas FtsA27 was defective in both activities. FtsA27 was also less able to
localize to the Z ring in vivo. To investigate the role of ATP transactions in
FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor
lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at
the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably,
suppressors outside of the ATP binding site, including some mapping to the
FtsA-FtsA subunit interface, also enhanced ATP transactions and exhibited gain
of function phenotypes in vivo. These results suggest that allosteric effects,
including changes in oligomeric state, may influence the ability of FtsA to bind
and/or hydrolyse ATP. Cell division in Chlamydiae is poorly understood as apparent homologs to most
conserved bacterial cell division proteins are lacking and presence of
elongation (rod shape) associated proteins indicate non-canonical mechanisms may
be employed. The rod-shape determining protein MreB has been proposed as playing
a unique role in chlamydial cell division. In other organisms, MreB is part of
an elongation complex that requires RodZ for proper function. A recent study
reported that the protein encoded by ORF CT009 interacts with MreB despite low
sequence similarity to RodZ. The studies herein expand on those observations
through protein structure, mutagenesis and cellular localization analyses.
Structural analysis indicated that CT009 shares high level of structural
similarity to RodZ, revealing the conserved orientation of two residues critical
for MreB interaction. Substitutions eliminated MreB protein interaction and
partial complementation provided by CT009 in RodZ deficient Escherichia coli.
Cellular localization analysis of CT009 showed uniform membrane staining in
Chlamydia. This was in contrast to the localization of MreB, which was
restricted to predicted septal planes. MreB localization to septal planes
provides direct experimental observation for the role of MreB in cell division
and supports the hypothesis that it serves as a functional replacement for FtsZ
in Chlamydia. The gram-positive bacterium Staphylococcus aureus, responsible for a wide
variety of diseases in human involve all organ systems ranging from localized
skin infections to life-threatening systemic infections. FtsZ, the key protein
of bacterial cell division was selected as a potent anti bacterial target. In
order to identify the new compounds structure based screening process was
carried out. An enrichment study was performed to select a suitable scoring
function and to retrieve potential candidates against FtsZ from a large chemical
database. The docking score and docking energy values were compared and their
atomic interaction was also evaluated. Furthermore molecular dynamics simulation
were also been performed to check the stability and the amino acids interacted
towards the FtsZ. Finally we selected C ID 16284, 25916, 15894, 13403 as better
lead compounds. From these results, we conclude that our insilico results will
provide a framework for the detailed in vitro and in vivo studies about the FtsZ
protein activity in drug development process. |
Intetumumab has been tested in clinical trials for treatment of which cancers? | Intetumumab has been tested in clinical trials for treatment of prostate cancer, melanoma and angiosarcoma. | Clinical and health-related quality of life (HRQoL) information was analyzed to
determine: (a) patient-reported signs, symptoms, and functioning, (b) HRQoL
questionnaire psychometrics, and (c) treatment impact on HRQoL. Data from the
Melanoma Subscale (MS) of the Functional Assessment of Cancer Therapy-Melanoma
and the worst pain question from the Brief Pain Inventory (BPI) were taken from
a clinical trial evaluating intetumumab alone or with dacarbazine in Stage IV
metastatic melanoma. Descriptive statistics examined patient-reported disease
burden at baseline. Correlations explored clinical endpoint and HRQoL
associations. Psychometrics included Cronbach's α internal consistency and
intraclass correlation coefficient (ICC). Treatment impact on HRQoL was
evaluated through HRQoL maintece and response analyses. Patients (n=127) had
a mean age of 62 years, a mean±SD hemoglobin of 13.0±2.6 g/dl, and a mean±SD
lactic dehydrogenase of 394±454 IU/l. Ninety-eight percent were Caucasian, 67%
were men, and 64% had an Eastern Cooperative Oncology Group status of 0.
Baseline BPI worst pain and MS scores (mean±SD) were 1.6±2.2 and 54.5±7.2,
respectively. Top three patient-reported health decrements in the MS were
appetite, fatigue, and limited physical activity. Observed HRQoL decrements were
consistent with the literature. MS and BPI worst pain item demonstrated good
psychometrics: Cronbach's α and ICC for the MS were 0.79 and 0.86, respectively;
BPI ICC was 0.74. A trend for HRQoL response was observed 3 weeks postbaseline
in the dacarbazine + 10 mg/kg intetumumab arm compared with dacarbazine +
placebo: 22 versus 10%, respectively, for the MS; 23 versus 5% for the BPI.
Further research on the HRQoL benefit of intetumumab in larger studies appears
warranted. BACKGROUND: Intetumumab is a fully human mAb with antiangiogenic, antitumor
properties which has shown potential therapeutic effect in castration-resistant
prostate cancer (CRPC) patients.
PATIENTS AND METHODS: In a phase 2, randomized, double-blind, multicenter study,
men with metastatic CRPC without prior systemic nonhormonal therapy were
randomly assigned to 75-mg/m(2) docetaxel (Taxotere) and 5-mg prednisone plus
placebo (N = 65) or 10-mg/kg intetumumab (N = 66) q3w. Placebo patients with
progressive disease (PD) could cross over to 10-mg/kg intetumumab alone or with
docetaxel. The primary end-point was progression-free survival (PFS). The
secondary end-points included tumor response (complete response + partial
response, CR + PR), prostate-specific antigen (PSA) response, and overall
survival (OS).
RESULTS: All efficacy end-points favored placebo over intetumumab, including PFS
(median 11.0 versus 7.6 months, P = 0.014), tumor response (20% versus 16%, P =
0.795), PSA response (68% versus 47%, P = 0.018), OS (median 20.6 versus 17.2
months, P = 0.163). Common all-grade adverse events (AEs) with placebo and
intetumumab were alopecia (43% versus 26%); diarrhea, leukopenia (both 34%
versus 27%); neutropenia (35% versus 23%). Grade ≥ 3 leukopenia (28% versus 17%)
and neutropenia (26% versus 18%) occurred more often with placebo than with
intetumumab. Intetumumab serum concentrations increased with repeated dosing and
did not reach steady-state. Greater decreases in N-telopeptide of type I
collagen (NTx), C-telopeptide (CTx) and CTCs occurred with intetumumab than with
placebo.
CONCLUSION: The addition of intetumumab to docetaxel resulted in shorter PFS
without additional toxicity among CRPC patients. |
Is there any evidence of dysregulated long non coding RNAs and Alzheimer's disease? | It is becoming increasingly evident that long non coding RNAs (lncRNAs) play a role on neurodegenerative diseases such as Alzheimer (AD). BACE1-AS, GDNFOS and 17A are examples of these lncRNAs. In some instances those lncRNAs are embedded or transcribed from the opposite strand of coding genes. | Alternative splicing is a central component of human brain complexity;
nonetheless, its regulatory mechanisms are still largely unclear. In this work,
we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol)
III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51,
GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells
induces the synthesis of an alternative splicing isoform that abolish GABA B2
intracellular signaling (i.e., inhibition of cAMP accumulation and activation of
K(+) channels). Indeed, 17A is expressed in human brain, and we report that it
is upregulated in cerebral tissues derived from Alzheimer disease patients. We
demonstrate that 17A expression in neuroblastoma cells enhances the secretion of
amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its
synthesis is induced in response to inflammatory stimuli. These data correlate,
for the first time, the activity of a novel pol III-dependent ncRNA to
alternative splicing events and, possibly, to neurodegeneration induced by
abnormal GABA B function. We anticipate that further analysis of pol
III-dependent regulation of alternative splicing will disclose novel regulatory
pathways associated to brain physiology and/or pathology. Primate-specific genes and isoforms could provide insight into human brain
diseases. Our bioinformatic analysis revealed that there are possibly five
isoforms of human GDNF gene with different pre- and pro-regions by inter- and
intra-exon splicing. By using TaqMan primer probe sets, designed between exons,
we verified the expression of all isoforms. Furthermore, a novel GDNFOS gene was
found to be transcribed from the opposite strand of GDNF gene. GDNFOS gene has
four exons that are spliced into different isoforms. GDNFOS1 and GDNFOS2 are
long noncoding RNAs, and GDNFOS3 encodes a protein of 105 amino acids. To study
human GDNF and GDNFOS regulation in neurodegenerative diseases, the protein and
mRNA levels were measured by Western blot and RT-quantitative PCR, respectively,
in postmortem middle temporal gyrus (MTG) of Alzheimer disease (AD) and
Huntington disease (HD) patients in comparison with those of normal controls. In
the MTG of AD patients, the mature GDNF peptide was down-regulated; however, the
transcript of GDNF isoform from human exon 2 was up-regulated, whereas that of
the conserved isoform from exon 1 remained unchanged in comparison with those of
normal controls. In contrast, the mature GDNF peptide and the isoform mRNA
levels were not changed in the MTG of HD. The findings of novel GDNF and GDNFOS
isoforms and differences in tissue expression patterns dysregulated in AD brains
may further reveal the role of endogenous GDNF in human brain diseases. Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene
for late-onset Alzheimer's disease (AD), although its role in the aetiology
and/or progression of this disorder is not fully understood. Here, we report the
finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in
antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a
splicing shift of SORL1 from the synthesis of the canonical long protein variant
A to an alternatively spliced protein form. This process, resulting in a
decreased synthesis of SORL1 variant A, is associated with impaired processing
of amyloid precursor protein (APP), leading to increased Aβ formation.
Interestingly, we found that 51A is expressed in human brains, being frequently
upregulated in cerebral cortices from individuals with Alzheimer's disease.
Altogether, these findings document a novel ncRNA-dependent regulatory pathway
that might have relevant implications in neurodegeneration. Long noncoding RNA (lncRNA) within mRNA sequences of Alzheimer's disease genes,
namely, APP, APOE, PSEN1, and PSEN2, has been analyzed using fractal dimension
(FD) computation and correlation analysis. We examined lncRNA by comparing mRNA
FD to corresponding coding DNA sequences (CDSs) FD. APP, APOE, and PSEN1 CDSs
select slightly higher FDs compared to the mRNA, while PSEN2 CDSs FDs are lower.
The correlation coefficient for these sequences is 0.969. A comparative study of
differentially expressed MAPK signaling pathway lncRNAs in pancreatic cancer
cells shows a correlation of 0.771. Selection of higher FD CDSs could indicate
interaction of Alzheimer's gene products APP, APOE, and PSEN1. Including
hypocretin sequences (where all CDSs have higher fractal dimensions than mRNA)
in the APP, APOE, and PSEN1 sequence analyses improves correlation, but the
inclusion of erythropoietin (where all CDSs have higher FD than mRNA) would
suppress correlation, suggesting that HCRT, a hypothalamus neurotransmitter
related to the wake/sleep cycle, might be better when compared to EPO, a
glycoprotein hormone, for targeting Alzheimer's disease drug development.
Fractal dimension and entropy correlation have provided supporting evidence,
consistent with evolutionary studies, for using a zebrafish model together with
a mouse model, in HCRT drug development. Long noncoding RNAs (lncRNAs) have been attracting immense research interest,
while only a handful of lncRNAs have been characterized thoroughly. Their
involvement in the fundamental cellular processes including regulate gene
expression at epigenetics, transcription, and post-transcription highlighted a
central role in cell homeostasis. However, lncRNAs studies are still at a
relatively early stage, their definition, conservation, functions, and action
mechanisms remain fairly complicated. Here, we give a systematic and
comprehensive summary of the existing knowledge of lncRNAs in order to provide a
better understanding of this new studying field. lncRNAs play important roles in
brain development, neuron function and maintece, and neurodegenerative
diseases are becoming increasingly evident. In this review, we also highlighted
recent studies related lncRNAs in central nervous system (CNS) development and
neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's
disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS),
and elucidated some specific lncRNAs which may be important for understanding
the pathophysiology of neurodegenerative diseases, also have the potential as
therapeutic targets. |
Which hormone receptor function is altered in patients with Donohue syndrome? | Donohue syndrome (leprechaunism) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene causing either defects in insulin binding or receptor autophosphorylation and tyrosine kinase activity. This syndrome is characterized by excessive hyperglycemia with hyperinsulinism, pre- and postnatal growth retardation, distinct dysmorphism and early death. Progressive obstructive cardiomyopathy and renal tubular dysfunction have been described in patients with Donohue syndrome. Milder form of insulin resistance due to insulin receptor gene mutation is coined as Rabson-Mendenhall syndrome. | OBJECTIVE/PATIENTS: Rabson-Mendenhall syndrome (RMS) is a rare, recessively
inherited disorder of extreme insulin resistance due to mutations in the insulin
receptor gene. We have identified a pair of siblings with RMS attributable to
compound heterozygosity for two insulin receptor mutations, one previously
unreported, and have characterized the novel receptor mutation functionally.
MEASUREMENTS: Insulin receptor sequencing was performed to identify the
mutations. Expression levels of the mature receptor were determined in
lymphoblastoid cells from the affected subjects. Further studies of immortalized
cell lines transfected with mutant and wild type (WT) receptors were undertaken
to characterize the effects of the novel mutation on [(125)I]-labelled insulin
binding, proreceptor processing and insulin-stimulated receptor
autophosphorylation.
RESULTS: Sequencing of the insulin proreceptor coding sequence revealed both
siblings to be compound heterozygotes for the missense mutations Arg209His and
Gly359Ser in the mature insulin receptor. The former mutation has been described
in homozygous form in Donohue syndrome, while the latter is novel. Insulin
receptor expression in lymphoblastoid cell lines was present at only 10-30% of
that in control cells; studies of immortalized cells transfected with mutant and
WT receptors confirmed the reduced expression of the mutant. The degree of
impairment of insulin binding and insulin-stimulated receptor
autophosphorylation were commensurate with the decrease in expression of the
mature receptor.
CONCLUSIONS: Loss of function of the novel insulin receptor (INSR) G359S variant
is largely accounted for by aberrant proreceptor processing rather than
intrinsically impaired signal transduction by the mutant receptor. Homozygous or compound heterozygous mutations within the insulin binding domain
of the human insulin receptor (INSR) are usually associated with severe
impairment of insulin binding leading to Donohue syndrome ("Leprechaunism"),
which is characterized by excessive hyperglycemia with hyperinsulinism, pre- and
postnatal growth retardation, distinct dysmorphism and early death. Missense
mutations in the beta subunits are commonly associated with a milder impairment
of insulin binding and milder phenotype with prolonged survival and less
dysmorphism, the so called Rabson-Mendenhall syndrome. We report on a
13-year-old girl with Donohue syndrome like dysmorphism, hyperinsulinism and
prolonged survival due to two novel INSR missense mutations within the insulin
binding domain. Unexpectedly, insulin binding assays and investigations of
activation of central insulin signaling pathways in fibroblasts revealed no
significant alterations. Instead, immunofluorescence studies showed abnormal
perinuclear distribution of the INSR alpha and beta subunits. Our data indicate
that the quality of insulin binding activity is correlated with survival, not
with the dysmorphic phenotype, and it is not always a valid parameter for
predicting INSR mutations as proposed. PURPOSE: Leprechaunism is a rare congenital syndrome caused by mutations of the
insulin receptor gene, transmitted in an autosomal recessive pattern. Insulin
growth factor-1 (IGF-1) treatment can be a therapeutic option in this syndrome
by its insulin-like effects. Nevertheless, it is of note that IGF-1 has also an
angiogenic activity.
METHODS: Fundus examination by ophthalmoscopy, fluorangiography, and laser
treatment were performed.
RESULTS: A 17-year-old girl with leprechaunism, under treatment with high doses
of insulin, presented a florid diabetic retinopathy. The large
neovascularization of the disk regressed after treatment with argon laser
panretinal photocoagulation. Five years after treatment, the patient maintained
good vision.
CONCLUSIONS: This clinical case is of interest for 2 reasons: 1) the large
retinal neovascularization was likely due to the high insulin dosages; 2) this
is the first case in which a sustained regression of retinal neovascularization
has been observed after laser treatment in leprechaunism. Leprechaunism (Donohue syndrome) is the most severe type of insulin receptor
(INSR) gene anomaly with the majority of patients surviving for only 2 years. We
report a surviving 2 -year-old male with leprechaunism, bearing novel compound
heterozygous mutations in the INSR. The patient is a Japanese boy with
acanthosis nigricans, lack of subcutaneous fat, hirsutism, thick lips, gum
hypertrophy and extremely high insulin levels (6702 mU/mL). He was as having
identified novel compound heterozygous mutations in INSR (p.T910M and p.
E1047K). At 24 day-old, recombit human insulin-like growth factor 1 (rh-IGF1)
treatment was started because of poor weight gain. At 2 years old, the patient's
serum glucose level and HbA1C value had worsened, and both a bolus of rh-IGF-1
and a subcutaneous injection of a rapid-acting insulin analog after meals, in
addition to α-glycosidase inhibitor, were initiated from 2 years onward. Oxygen
administration and biphasic positive airway pressure treatment were also
initiated from 2 years old due to upper airway obstruction with adenoidal
hypertrophy. In the experiments conducted using COS7 cells homozygously
transfected with the INSR mutation, T910M INSR failed to process the proreceptor
and decreased insulin-stimulated tyrosine phosphorylation. E1047K INSR resulted
in a complete absence of insulin-stimulated tyrosine phosphorylation. These
findings suggest the near absence of INSR in this patient. We consider that the
rhIGF1 treatment contributed to his long survival, but it was not able to
prevent his diabetic condition. Our report provides important insights into the
function of INSR, and for the treatment of leprechaunism. Donohue syndrome (leprechaunism; OMIM *246200) is a rare, recessively inherited
disorder of extreme insulin resistance due to mutations in the insulin receptor
gene (INSR) causing either defects in insulin binding or receptor
autophosphorylation and tyrosine kinase activity. We report a patient with
pronounced clinical picture of leprechaunism who developed severe progressive
hypertrophic obstructive cardiomyopathy (HOCM) and renal tubular dysfunction
which improved on continuous subcutaneous infusion of recombit human
insulin-like growth factor-1 (rhIGF-I). INSR gene molecular analysis and insulin
receptor (IR) autophosphorylation on cultured fibroblasts were performed. A
novel homozygous missense mutation p.Leu795Pro was found, located in the
extracellular portion of the β subunit of the insulin receptor. The post-binding
defect of the insulin receptor signaling in cultured fibroblasts demonstrated
decreased insulin receptor autophosphorylation.
CONCLUSION: Treatment with rhIGF-I partially reversed severe progressive HOCM
and renal tubular dysfunction in a patient with Donohue syndrome associated with
a novel p.Leu795Pro INSR gene mutation causing a severe decrease in IR
autophosphorylation. Leprechaunism is a rare autosomal recessive disease that is characterized by
severe insulin resistance. This disease is caused by a defective insulin
receptor and features abnormal glucose metabolism and retarded intrauterine and
postnatal growth. However, there are few reports on the long-term course of
leprechaunism. We reported the long-term clinical course and rh-IGF-1 treatment
in a patient with leprechaunism. During follow-up her diabetes gradually
deteriorated despite of treatment of rh-IGF-1. Furthermore, she developed
endometrioid adenocarcinoma at the age of 24 yr. The development of endometrial
disease must be carefully followed up in this disease. |
Does triiodothyronine (T3) has cardiac angiogenic effects? | T3-induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF-BB, PDGFR-β and downstream activation of Akt.
T(3) administration restored TRbeta mRNA expression level in AAC hearts to the control level.
TRbeta in the coronary ECs regulates capillary density during cardiac development, and down-regulation of TRbeta results in coronary microvascular rarefaction during pathological hypertrophy. | The effects of thyroxine-stimulated hypertrophy (TSH) were studied in the
porcine left ventricular myocardium. Hypertrophy was produced in six adult pigs
by administration of triiodothyronine (1 mg/kg; i.v.) for eight days. Six pigs
served as controls. The degree of hypertrophy, determined by left
ventricular-to-body weight ratio, was 47%. With hypertrophy there was a
significant increase in heart rate, blood pressure and myocardial blood flows.
Minimal coronary resistance measured during adenosine infusion was lower in the
TSH group compared with the control group. Anatomic studies revealed a balanced
proliferative response of mitochondria, myofibrils and the t-tubular system
during TSH. Analysis of the microvasculature indicated that the capillary and
arteriolar beds both experienced growth which paralleled myocyte growth during
TSH. These results suggest that thyroxine administration promotes angiogenesis
in the microvascular bed which provides a partial anatomic rationale for the
lowered minimal coronary resistance. Insufficient angiogenesis is one of the causes leading to tissue ischemia and
dysfunction. In heart failure, there is increasing evidence showing decreased
capillary density in the left ventricle (LV) myocardium, although the detailed
mechanisms contributing to it are not clear. The goal of this study was to
investigate the role of thyroid hormone receptors (TRs) in the coronary
microvascular rarefaction under pathological cardiac hypertrophy. The LV from
hypertrophied/failing hearts induced by ascending aortic constriction (AAC)
exhibited severe microvascular rarefaction, and this phenomenon was restored by
chronic T(3) administration. Coronary endothelial cells (ECs) isolated from AAC
hearts expressed lower TRbeta mRNA than control ECs, and chronic T(3)
administration restored TRbeta mRNA expression level in AAC hearts to the
control level. Among different TR subtype-specific knockout mice, TRbeta
knockout and TRalpha/TRbeta double-knockout mice both exhibited significantly
less capillary density in LV compared with wild-type mice. In vitro, coronary
ECs isolated from TRbeta knockout mice lacked the ability to form capillary
networks. In addition, we identified that kinase insert domain protein
receptor/fetal liver kinase-1 (vascular endothelial growth factor-2 receptor)
was one of the angiogenic mediators controlled by T(3) administration in the AAC
heart. These data suggest that TRbeta in the coronary ECs regulates capillary
density during cardiac development, and down-regulation of TRbeta results in
coronary microvascular rarefaction during pathological hypertrophy. Patients with hypothyroidism are at a higher risk for coronary vascular disease.
Patients with diabetes and related vascular complications also have an increased
incidence of low thyroid function. While thyroid hormones (THs) may be key
regulators of a healthy vasculature, potential undesirable side effects hinder
their use in the treatment of vascular disorders. TH analogs such as
3,5-diiodothyropropionic acid (DITPA) may provide a safer treatment option.
However, the relative potency of DITPA on vascular growth, cardiac function, and
metabolism is poorly understood. We hypothesized that the vascular
growth-promoting effects of DITPA can be obtained with a minimum effect on
cardiac function. Thyroidectomized Sprague-Dawley rats were given slow-release
pellets with either thyroxine (T4, 2.7 or 5.2 mg) or DITPA (80 mg) for 6 wk and
were compared with placebo. Heart mass, body mass, body temperature, serum THs,
cardiac function (echocardiograms and hemodynamics), and myocardial arteriolar
density were determined. Hypothyroidism led to reductions in cardiac function,
heart mass, body temperature, and myocardial arterioles. High-dose T4 prevented
arteriolar loss and the development of hypothyroidism. Low-dose T4 partially
prevented the reduction in cardiac function but had minimal effects on
arteriolar loss. In contrast, DITPA treatment prevented myocardial arteriolar
loss but not the progression of hypothyroid-induced changes in cardiac function.
The results suggested that DITPA can promote a healthy vasculature independently
from its thyroid-related metabolic effects. Drugs in this class may provide new
therapeutic options for patients with vascular disease. 3,5,3'-Levo-triiodothyronine (L-T3) is essential for DNA transcription,
mitochondrial biogenesis and respiration, but its circulating levels rapidly
decrease after myocardial infarction (MI). The main aim of our study was to test
whether an early and sustained normalization of L-T3 serum levels after MI
exerts myocardial protective effects through a mitochondrial preservation.
Seventy-two hours after MI induced by anterior interventricular artery ligation,
rats were infused with synthetic L-T3 (1.2 μg/kg/day) or saline over 4 weeks.
Compared to saline, L-T3 infusion restored FT3 serum levels at euthyroid state
(3.0 ± 0.2 versus 4.2 ± 0.3 pg/ml), improved left ventricular (LV) ejection
fraction (39.5 ± 2.5 versus 65.5 ± 6.9%), preserved LV end-systolic wall
thickening in the peri-infarct zone (6.34 ± 3.1 versus 33.7 ± 6.21%) and reduced
LV infarct-scar size by approximately 50% (all P < 0.05). Moreover, L-T3
significantly increased angiogenesis and cell survival and enhanced the
expression of nuclear-encoded transcription factors involved in these processes.
Finally, L-T3 significantly increased the expression of factors involved in
mitochondrial DNA transcription and biogenesis, such as hypoxic inducible
factor-1α, mitochondrial transcription factor A and peroxisome proliferator
activated receptor γ coactivator-1α, in the LV peri-infarct zone. To further
explore mechanisms of L-T3 protective effects, we exposed isolated neonatal
cardiomyocytes to H(2)O(2) and found that L-T3 rescued mitochondrial biogenesis
and function and protected against cell death via a mitoKATP dependent pathway.
Early and sustained physiological restoration of circulating L-T3 levels after
MI halves infarct scar size and prevents the progression towards heart failure.
This beneficial effect is likely due to enhanced capillary formation and
mitochondrial protection. Study of physiological angiogenesis and associated signalling mechanisms in
adult heart has been limited by the lack of a robust animal model. We
investigated thyroid hormone-induced sprouting angiogenesis and the underlying
mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with
propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12
weeks- (young) and 1 year-PTU (middle age) treatment caused a remarkable
capillary rarefaction observed in capillary density. Three-day Triiodothyronine
(T3) treatment significantly induced cardiac capillary growth in hypothyroid
mice. In cultured left ventricle (LV) tissues from PTU-treated mice, T3 also
induced robust sprouting angiogenesis where pericyte-wrapped endothelial cells
formed tubes. The in vitro T3 angiogenic response was similar in mice
pre-treated with PTU for periods ranging from 1.5 to 12 months. Besides bFGF and
VEGF(164) , PDGF-BB was the most robust angiogenic growth factor, which
stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues
with increasing potency, but had little effect on tissues from euthyroid mice.
T3 treatment significantly increased PDGF receptor beta (PDGFR-β) protein levels
in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on
sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. In
addition, activation of Akt signalling mediated in T3-induced angiogenesis was
blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that
hypothyroidism leads to cardiac microvascular impairment and rarefaction with
increased sensitivity to angiogenic growth factors. T3-induced cardiac sprouting
angiogenesis in adult hypothyroid mice was associated with PDGF-BB, PDGFR-β and
downstream activation of Akt. |
Is the HRC Ser96Ala variant associated with sudden cardiac death in patients with dilated cardiomyopathy? | A human genetic variant (Ser96Ala) in the sarcoplasmic reticulum (SR) histidine-rich Ca(2+)-binding (HRC) protein has been linked to ventricular arrhythmia and sudden death in dilated cardiomyopathy.The histidine-rich calcium binding protein (HRC) Ser96Ala polymorphism was shown to correlate with ventricular arrhythmias and sudden death only in dilated cardiomyopathy patients but not in healthy human carriers. | AIMS: To investigate whether genetic variants of the histidine-rich calcium
(HRC)-binding protein are associated with idiopathic dilated cardiomyopathy
(DCM) and its progression.
METHODS AND RESULTS: We screened 123 idiopathic DCM patients and 96 healthy
individuals by single-strand conformation polymorphism analysis and direct
sequencing for genetic variants in HRC. Six polymorphisms were detected:
Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (-/GAG),
Asp261del (GAT/-), and an in-frame insertion of 51 amino acids at His321. The
analysis of their frequencies did not reveal any significant correlation with
DCM development. However, the Ser96Ala polymorphism exhibited a statistically
significant correlation with the occurrence of life-threatening ventricular
arrhythmias. During a follow-up of 4.02 +/- 2.4 years, the risk for ventricular
arrhythmias was higher (HR, 9.620; 95% CI, 2.183-42.394; P = 0.003) in the
Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable
Cox regression analysis, the Ser96Ala polymorphism was the only significant
genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI,
0.838-20.967; P = 0.018).
CONCLUSION: The Ser96Ala genetic variant of HRC is associated with
life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an
independent predictor of susceptibility to arrhythmogenesis in the setting of
DCM. BACKGROUND: A human genetic variant (Ser96Ala) in the sarcoplasmic reticulum
(SR) histidine-rich Ca(2+)-binding (HRC) protein has been linked to ventricular
arrhythmia and sudden death in dilated cardiomyopathy. However, the precise
mechanisms affecting SR function and leading to arrhythmias remain elusive.
METHODS AND RESULTS: We generated transgenic mice with cardiac-specific
expression of human Ala96 HRC or Ser96 HRC in the null background to assess
function in absence of endogenous protein. Ala96 HRC decreased (25% to 30%)
cardiomyocyte contractility and Ca2+ kinetics compared with Ser96 HRC in the
absence of any structural or histological abnormalities. Furthermore, the
frequency of Ca2+ waves was significantly higher (10-fold), although SR Ca2+
load was reduced (by 27%) in Ala96 HRC cells. The underlying mechanisms involved
diminished interaction of Ala96 HRC with triadin, affecting ryanodine receptor
(RyR) stability. Indeed, the open probability of RyR, assessed by use of
ryanodine binding, was significantly increased. Accordingly, stress conditions
(5 Hz plus isoproterenol) induced aftercontractions (65% in Ala96 versus 12% in
Ser96) and delayed afterdepolarizations (70% in Ala96 versus 20% in Ser96). The
increased SR Ca2+ leak was accompanied by hyperphosphorylation (1.6-fold) of RyR
at Ser2814 by calmodulin-dependent protein kinase II. Accordingly, inclusion of
the calmodulin-dependent protein kinase II inhibitor KN93 prevented Ser2814
phosphorylation and partially reversed the increases in Ca2+ spark frequency and
wave production. Parallel in vivo studies revealed ventricular ectopy on
short-term isoproterenol challenge and increased (4-fold) propensity to
arrhythmias, including nonsustained ventricular tachycardia, after myocardial
infarction in Ala96 HRC mice.
CONCLUSIONS: These findings suggest that aberrant SR Ca2+ release and increased
susceptibility to delayed afterdepolarizations underlie triggered arrhythmic
activity in human Ala96 HRC carriers. The Ser96Ala (S96A) mutation within the histidine rich Ca(2+) binding protein
(HRC) has recently been linked to cardiac arrhythmias in idiopathic dilated
cardiomyopathy patients, potentially attributable to an increase in spontaneous
Ca(2+) release events. However, the molecular mechanism connecting the S96A
mutation of HRC to increased Ca(2+) release events remains unclear. Previous
findings by our group indicate that these spontaneous Ca(2+) release events may
be linked to store overload induced Ca(2+) release (SOICR) via the cardiac
ryanodine receptor (RyR2). Therefore, in the present study we sought to
determine whether HRC wild type (HRC WT) and S96A mutant (HRC S96A) expression
has a direct effect on SOICR. Using both cytosolic and intra-Ca(2+) store
measurements in human embryonic kidney cells expressing RyR2, we found that HRC
WT significantly inhibited the propensity for SOICR by buffering store free
Ca(2+) and inhibiting store Ca(2+) uptake. In contrast, HRC S96A exhibited a
markedly suppressed inhibitory effect on SOICR, which was attributed to an
impaired ability to buffer store Ca(2+) and reduce store Ca(2+) uptake. In
addition to impairing the ability of HRC to regulate bulk store Ca(2+), a
proximity ligation assay demonstrated that the S96A mutation also disrupts the
Ca(2+) microdomain around the RyR2, as it alters the Ca(2+) dependent
association of RyR2 and HRC. Importantly, in contrast to previous reports, the
absence of triadin in our experimental model illustrates that the S96A mutation
in HRC can alter the propensity for SOICR without any interaction with triadin.
Collectively, our results demonstrate that the human HRC mutation S96A leads to
an increase in spontaneous Ca(2+) release and ultimately arrhythmias by
disrupting the regulation of intra-store free Ca(2+). This is primarily due to
an impaired ability to act as an effective bulk and local microdomain store
Ca(2+) buffer. |
Are BRAF mutations common in melanoma? | Melanoma is the most aggressive form of skin cancer. The treatment of patients with advanced melanoma is rapidly evolving due to an improved understanding of molecular drivers of this disease. Somatic mutations in BRAF are the most common genetic alteration found in these tumors. BRAF mutations occur in approximately 8% of all human cancers and approach 50% in melanoma and papillary carcinoma of thyroid. | PURPOSE: Recently, it was reported that BRAF mutations are frequent in melanoma.
Previously, we analyzed a large series of paired primary and metastatic
melanomas for NRAS codon 61 mutations and showed that they arise early and are
preserved during tumor progression. Here, we have screened the same tumor
samples for BRAF mutations.
EXPERIMENTAL DESIGN: Primary melanomas (n = 71) and corresponding metastases (n
= 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations
using single-strand conformational polymorphism and nucleotide sequence analysis
RESULTS: BRAF mutations were found in 42 of 71 patients (59%). Thirty-seven
patients had mutations that lead to a Val599Glu change, whereas mutations
resulting in Gly468Ser, Val599Arg, Val599Lys, and Lys600Glu changes were
detected in one patient each. Furthermore, one patient had a 6-bp insertion
between codons 598 and 599, encoding two threonine residues. In most cases,
paired primary and metastatic lesions had the same BRAF genotype (i.e.,
mutations present in the primary tumors were preserved in the corresponding
metastases, and mutations did not arise at the metastatic stage if they were not
present in the primary lesion). Using laser-capture microdissection, BRAF
mutations were found in the radial growth phase of the primary lesions. BRAF
mutations occurred exclusively in tumors that were wild type for NRAS, and in
total, 89% of the patients analyzed (63 of 71) had mutations in either of these
two genes.
CONCLUSIONS: The Ras-Raf-mitogen-activated protein kinase/extracellular
signal-regulated kinase-extracellular signal-regulated kinase signaling pathway
is activated in the vast majority of melanomas. Activation occurs through either
NRAS or BRAF mutations, both of which arise early during melanoma pathogenesis
and are preserved throughout tumor progression. BACKGROUND: Maligt melanoma arising from different body compartments may be
associated with differing aetiological factors and clinical behaviour, and may
manifest diverse molecular genetic profiles. Although many studies have focused
on cutaneous melanoma, little is known of mucosal and other types of melanoma.
In particular, maligt melanoma of soft parts is different from other
melanomas in many respects, yet manifests a common melanocytic differentiation.
Mutation of BRAF is now known to be common in cutaneous melanomas, and raises
possible new therapeutic options of anti-RAF treatment for these patients. Few
data are available for non-cutaneous melanomas.
AIMS: To study the incidence of BRAF and NRAS mutations in melanomas arising in
diverse internal organs.
METHODS: Fifty one melanomas from various internal organs were investigated for
BRAF and NRAS mutation by direct DNA sequencing.
RESULTS: BRAF and NRAS mutations were found in two and five mucosal melanomas
arising from the aerodigestive and female genital tracts (n = 36). Their
occurrence is mutually exclusive, giving a combined mutation incidence rate of
19.4% in mucosal melanomas. Both BRAF and NRAS mutations were absent in
maligt melanoma of soft parts (n = 7). BRAF mutation was also absent in uveal
melanoma (n = 6), but was seen in two of five cutaneous melanomas. The incidence
of BRAF or combined BRAF/NRAS mutations in all non-cutaneous groups was
significantly lower than published rates for cutaneous melanomas.
CONCLUSION: Each melanoma subtype may have a unique oncogenetic pathway of
tumour development, and only a small fraction of non-cutaneous melanomas may
benefit from anti-RAF treatment. BRAF mutations are common events in a variety of melanocytic nevi and primary
cutaneous melanomas. We have previously found BRAF mutations in 82% of nevi,
consisting of congenital, common acquired and dysplastic types, and 33% of
primary cutaneous melanomas other than the spitzoid type, similar to other
published reports. A small number of studies have evaluated Spitz nevi and have
failed to detect any lesions possessing a BRAF mutation. Only one study included
categories of atypical Spitz nevus and borderline lesions suspected to be
spitzoid melanomas, along with classic Spitz nevi and spitzoid melanomas. We
examined a spectrum of spitzoid lesions that included 48 Spitz nevi, some with
atypical features, seven atypical (borderline) Spitz tumors, and 13 spitzoid
melanomas. BRAF mutations were detected in 12 of 68 spitzoid lesions, of which
two were spitzoid melanomas and 10 were Spitz nevi. Five of the 10 Spitz nevi
with BRAF mutations were altered by more than usual cytologic atypia and/or
architectural atypia overlapping with dysplastic nevi, or
irritation/inflammation; one desmoplastic Spitz nevus had a BRAF mutation. These
results indicate that a small subset of Spitz nevi, some with atypical
histologic features, possess BRAF mutations. Therefore, the BRAF mutational
status does not separate all Spitz nevi from spitzoid melanomas and non-Spitz
types of melanocytic proliferations, contrary to previous reports. Oncogenic BRAF and NRAS mutations are frequent in maligt melanoma. BRAF that
is activated by the common V600E and other mutations, as well as by upstream
NRAS mutations, has been shown to require the molecular chaperone heat shock
protein 90 (HSP90) for stabilization and is depleted by the HSP90 inhibitor
17-allylamino-17-demethoxygeldanamycin (17-AAG)]. Here, we explore the possible
relationship between tumor BRAF and NRAS mutations and clinical response to
17-AAG in six patients with metastatic maligt melanoma who received
pharmacologically active doses of 17-AAG as part of a phase I clinical trial.
One patient with disease stabilization for 49 months had a (G13D)NRAS mutation
and (WT)BRAF. A second patient who had stable disease for 15 months had a
(V600E)BRAF mutation and (WT)NRAS. These preliminary results suggest that BRAF
and NRAS mutation status should be determined in prospective phase II studies of
HSP90 inhibitors in melanoma. AIM: Approximately 40-60% of melanomas from Caucasian populations carry
activating mutations in the BRAF oncogene, with the most common being the
p.Val600Glu (V600E) hotspot mutation in exon 15. The aim of the present study
was to investigate the frequency of the less common p.Val600Lys (V600K) mutation
in metastatic melanoma from a high incidence region.
METHOD: Dideoxy sequencing and fluorescent single strand conformation analysis
were used to screen for mutations in exon 15 of BRAF in 183 cases of metastatic
melanoma.
RESULTS: The overall incidence of BRAF mutation (89/183, 49%) was very similar
to other large studies of Caucasian populations. However, the frequency of the
p.Val600Lys mutation was higher than in most other studies and comprised almost
one-third of all BRAF mutations in our cohort (27/89, 30%).
CONCLUSION: BRAF p.Val600Lys mutations were present at a relatively high
frequency in this cohort of metastatic melanoma patients (27/183, 15%). Assays
used to screen for BRAF mutations in the clinic should be robust enough to
detect the p.Val600Lys mutation, as this may have therapeutic implications. Activating mutations in the BRAF gene occur in approximately 50% of melanomas.
More than 70% of BRAF mutations are V600E and 10-30% are V600K. Potent and
selective BRAF inhibitors have demonstrated significant clinical benefits in
patients with V600E and V600K BRAF-mutated melanoma. V600R mutations constitute
approximately 3-7% of all BRAF mutations and the activity of BRAF inhibitors in
patients with this mutation is unknown. We have treated 45 patients with V600
mutated melanoma including patients with V600R mutation between July 2011 and
October 2012 with the selective BRAF inhibitor dabrafenib (n=43) or vemurafenib
(n=2) via a compassionate access programme. The overall response rate was 50%
for the whole population with a progression-free survival of 5.5 months. Five
objective responses were seen in six assessable patients with V600R BRAF
mutation (n=9). Our experience suggests that patients with V600R BRAF mutations
can be treated successfully with oral BRAF inhibitors, and molecular diagnostic
assays should include detection of this type of mutation. The US Food and Drug Administration (FDA) approved vemurafenib to treat patients
with metastatic melanoma harboring the BRAF c.1799T>A (p.V600E) mutation.
However, a subset of melanomas harbor non-p.V600E BRAF mutations, and these data
are of potential importance regarding the efficacy of current targeted
therapies. To better understand the BRAF mutation profile in melanomas, we
retrospectively analyzed data from 1112 primary and metastatic melanomas at our
institution. The cohort included nonacral cutaneous (n = 774), acral (n = 111),
mucosal (n = 26), uveal (n = 23), leptomeningeal (n = 1), and metastatic
melanomas of unknown primary site (n = 177). BRAF mutation hotspot regions in
exons 11 and 15 were analyzed by pyrosequencing or with the primer extension
MassARRAY system. A total of 499 (44.9%) specimens exhibited BRAF mutations,
involving exon 15 [497 (99.6%)] or exon 11 [2 (0.4%)]. p.V600E was detected in
376 (75.4%) cases; the remaining 123 (24.6%) cases exhibited non-p.V600E
mutations, of which p.V600K was most frequent [86 (17.2%)]. BRAF mutations were
more frequent in nonacral cutaneous (51.4%) than acral melanomas [18 (16.2%)] (P
< 0.001); however, there was no significant difference among cutaneous
histological subtypes. All mucosal, uveal, and leptomeningeal melanomas were
BRAF wild type (WT). The high frequency of non-p.V600E BRAF mutations in
melanoma has important implications because the FDA-approved companion
diagnostic test for p.V600E detects some but not all non-p.V600E mutations.
However, the therapeutic efficacy of vemurafenib is not well established in
these lesions. BRAF mutations have been identified as the most common oncogene mutation in
melanomas, especially important in those originating on nonchronically
sun-damaged skin. There is a large and continually growing body of evidence
regarding the importance of this mutation in targeted therapy for melanoma. In
this review, we outline these findings including: molecular pathways used by
BRAF, the importance in nonmaligt neoplasms, histologic associations, the
relationship of BRAF to KIT and NRAS mutations, and their impact on survival, as
well as resistance mechanisms to BRAF inhibitors employed by melanoma.
Understanding these topics and how they relate to one another may facilitate the
development of new treatments and eventually improve the prognosis for those
patients afflicted with this disease. Melanoma is an aggressive form of skin cancer that causes the greatest number of
skin cancer-related deaths worldwide. In its early stages maligt melanoma can
be cured by surgical resection, but once it has progressed to the metastatic
stage it is extremely difficult to treat and does not respond to current
therapies. A majority of cutaneous melanomas show activating mutations in the
NRAS or BRAF proto-oncogenes, components of the Ras-Raf-Mek-Erk (MAPK) signal
transduction pathway. The discovery of activating BRAF mutations in ∼50% of all
melanomas has proved to be a turning point in the therapeutic management of the
disseminated disease. This review summarizes the critical role of BRAF in
melanoma pathophysiology, the clinical and pathological determits of BRAF
mutation status and finally addresses the current state of the art of BRAF
inhibitors. We further outline the most recent findings on the mechanisms that
underlie intrinsic and acquired BRAF inhibitor resistance and describe ongoing
preclinical and clinical studies designed to delay or abrogate the onset of
therapeutic escape. Activating mutations in BRAF are the most common genetic alterations in
melanoma. Inhibition of BRAF by small molecules leads to cell-cycle arrest and
apoptosis. We show here that BRAF inhibition also induces an oxidative
phosphorylation gene program, mitochondrial biogenesis, and the increased
expression of the mitochondrial master regulator, PGC1α. We further show that a
target of BRAF, the melanocyte lineage factor MITF, directly regulates the
expression of PGC1α. Melanomas with activation of the BRAF/MAPK pathway have
suppressed levels of MITF and PGC1α and decreased oxidative metabolism.
Conversely, treatment of BRAF-mutated melanomas with BRAF inhibitors renders
them addicted to oxidative phosphorylation. Our data thus identify an adaptive
metabolic program that limits the efficacy of BRAF inhibitors. BRAF represents one of the most frequently mutated protein kinase genes in human
tumours. The mutation is commonly tested in pathology practice. BRAF mutation is
seen in melanoma, papillary thyroid carcinoma (including papillary thyroid
carcinoma arising from ovarian teratoma), ovarian serous tumours, colorectal
carcinoma, gliomas, hepatobiliary carcinomas and hairy cell leukaemia. In these
cancers, various genetic aberrations of the BRAF proto-oncogene, such as
different point mutations and chromosomal rearrangements, have been reported.
The most common mutation, BRAF V600E, can be detected by DNA sequencing and
immunohistochemistry on formalin fixed, paraffin embedded tumour tissue.
Detection of BRAF V600E mutation has the potential for clinical use as a
diagnostic and prognostic marker. In addition, a great deal of research effort
has been spent in strategies inhibiting its activity. Indeed, recent clinical
trials involving BRAF selective inhibitors exhibited promising response rates in
metastatic melanoma patients. Clinical trials are underway for other cancers.
However, cutaneous side effects of treatment have been reported and therapeutic
response to cancer is short-lived due to the emergence of several resistance
mechanisms. In this review, we give an update on the clinical pathological
relevance of BRAF mutation in cancer. It is hoped that the review will enhance
the direction of future research and assist in more effective use of the
knowledge of BRAF mutation in clinical practice. BRAF is the most prevalent oncogene and an important therapeutic target in
melanoma. In some cancers, BRAF is activated by rearrangements that fuse its
kinase domain to 5' partner genes. We examined 848 comparative genomic
hybridization profiles of melanocytic tumors and found copy number transitions
within BRAF in 10 tumors, of which six could be further characterized by
sequencing. In all, the BRAF kinase domain was fused in-frame to six N-terminal
partners. No other mutations were identified in melanoma oncogenes. One of the
seven melanoma cell lines without known oncogenic mutations harbored a similar
BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib,
but not vemurafenib, could block MAP kinase pathway activation and proliferation
of the cell line at clinically relevant concentrations, whereas BRAF(V) (600E)
mutant melanoma cell lines were significantly more sensitive to vemurafenib. The
patient from whom the cell line was derived showed a durable clinical response
to sorafenib. BACKGROUND: Treatment of advanced melanoma has been improved with the advent of
the BRAF inhibitors. However, a limitation to such treatment is the occurrence
of resistance. Several mechanisms have been identified to be responsible for the
development of resistance, either MEK-dependent or MEK-independent. In order to
overcome resistance due to reactivation of MEK signaling, MEK inhibitors are
being clinically developed with promising results. However, also in this case
resistance inevitably occurs. It has been recently reported that ErbB3, a member
of the EGFR receptor family, may be involved in the establishment of drug
resistance.
METHODS: Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L
(BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine
Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed
on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2
antibodies. The expression of neuregulin after vemurafenib treatment was
assessed by Real Time PCR and Western blotting. The growth inhibitory effects of
vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro
colony formation assays.
RESULTS: In the present study we demonstrate that ErbB3 is the main RTK
undergoing rapidly hyperphosphorylation upon either treatment with a BRAF
inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a
variety of V600BRAF mutations and that this results in a strong activation of
phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the
simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to
potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK
pathways and in the inhibition of melanoma cell growth. We show that
upregulation of phospho-ErbB3 is due to an autocrine loop involving increased
transcription and production of neuregulin by melanoma cells.
CONCLUSIONS: On the basis of these results, we propose that initial co-treatment
with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a
strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance
duration of clinical response. Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients
present with metastatic disease without an evident primary site. To determine
their likely site of origin, we combined exome sequencing from 33 MUPs to assess
the total rate of somatic mutations and degree of UV mutagenesis. An independent
cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly
prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed
that the majority of MUPs exhibited high somatic mutation rates, high ratios of
C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of
101, 32%) mutations, collectively indicating a mutation profile consistent with
cutaneous sun-exposed melanomas. These data suggest that a significant
proportion of MUPs arise from regressed or unrecognized primary cutaneous
melanomas or arise de novo in lymph nodes from nevus cells that have migrated
from the skin. The RAS/RAF/MEK/ERK pathway has been reported to be activated in over 80% of all
cutaneous melanomas, making it the focus of many scientific studies in the
melanoma field. Discoveries of mutations and aberrant expression of components
in this cascade, in particular, BRAF and NRAS render a deeper understanding of
the mechanisms responsible for oncogenesis and provide new therapeutic
strategies for this deadly disease. This review starts with a comprehensive
discussion on the role of this pathway in initiation and progress of melanoma.
Mechanistically, mutated BRAF and NRAS exert most of the oncogenic effects
through the activation of the MAPK pathway, which both drive the uncontrolled
growth of melanoma cells and regulate the cell survival. In a subsequent
section, clinical efficacy of targeted small-molecule inhibitors is highlighted.
BRAF-targeted therapies (e.g., vemurafenib, dabrafenib) have showed impressive
results in systemic therapy for melanoma harboring activating BRAF V600E
mutations. MEK inhibitors show limited activity in phase I trials, and
inhibitors directly targeting mutated NRAS, to date, have not been realized.
Furthermore, the emerging mechanisms underlying both intrinsic and acquired drug
resistance as well as approaches to prevent or abrogate the onset of therapeutic
escape are addressed. Finally, the promising vistas and major challenges
involving small-molecule inhibitors targeting this MAPK pathway in melanoma
therapy are briefly discussed. It can be envisaged that disseminated melanoma is
no longer such a bleak prognosis in future given the research and development of
new signal transduction inhibitors based on our evolving understanding of
melanoma genetics and intracellular signaling. Recent advances in molecular targeted therapies have greatly improved treatment
outcomes for cancers driven by oncogenic mutations. Despite initial and dramatic
clinical responses, tumors eventually acquire resistance to these targeted
therapies, showing flexible and diverse responses. Interestingly, cancer cells
sometimes overadapt to the drug treatment environment, leading to a state in
which cancer cells cannot survive without the drug. This interesting phenomenon
(often called "drug dependency" or "drug addiction") is exemplified in
preclinical acquired resistance models of BRAF-mutated melanoma treated with
vemurafenib and EGFR-mutated lung cancer treated with EGFR tyrosine kinase
inhibitors. A number of intriguing parallels in drug-addicted cancers became
apparent in a comparison of the two models: (i) overexpression of driver
oncogenes as causes of acquired resistance; (ii) overexpression of driver
oncogenes causing MEK-ERK hyperactivation under drug-free conditions; (iii)
hyperactivation of the MEK-ERK pathway as critical to this drug addiction
phenomenon; (iv) ongoing dependence on the oncogenic driver; and (v) morphologic
changes in resistant cells under drug-free conditions. This Perspective article
not only focuses on this interesting and peculiar phenomenon but also discusses
weapon strategies to exploit this unintentional weakness of cancers. RAF and MEK (mitogen-activated or extracellular signal-regulated protein kinase
kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma.
However, most responses are partial and short-lived, and many patients fail to
respond at all. We found that suppression of TORC1 activity in response to RAF
or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein
S6 (P-S6), effectively predicted induction of cell death by the inhibitor in
BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was
maintained after treatment with RAF or MEK inhibitors, in some cases despite
robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in
vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for
induction of apoptosis and tumor response. Finally, in paired biopsies obtained
from patients with BRAF-mutant melanoma before treatment and after initiation of
RAF inhibitor therapy, P-S6 suppression predicted significantly improved
progression-free survival. Such a change in P-S6 could be readily monitored in
real time by serial fine-needle aspiration biopsies, making quantitation of P-S6
a valuable biomarker to guide treatment in BRAF-mutant melanoma. INTRODUCTION: The clinical activity of BRAF inhibitor (BRAF-I) therapy is a
major breakthrough in the treatment of metastatic melanoma carrying BRAF
mutations. However, the therapeutic efficacy of BRAF-I therapy is limited due to
the onset of intrinsic and acquired drug resistance.
AREAS COVERED: The role of wild-type BRAF in melanocytes and of the mutated BRAF
in the pathogenesis of melanoma is described in this article. The results
obtained with BRAF-I in patients with mutated BRAF are reviewed. The mechanisms
driving the intrinsic and acquired BRAF-I resistance, the development of
combinatorial strategies designed to overcome them and their potential
limitations are discussed. Lastly, the many questions that have to be addressed
to optimize therapy with BRAF-I are listed.
EXPERT OPINION: Melanoma is an aggressive form of skin cancer characterized by
poor prognosis and high mortality. The discovery of BRAF mutations which drive
melanoma tumorigenesis and the development of agents which selectively inhibit
mutant-activated BRAF represent a major breakthrough in the treatment of
metastatic melanoma. However, the development of drug resistance underlies the
need of more effective and individualized combinatorial treatments to counteract
the multiple escape mechanisms utilized by BRAF-mutant melanoma. Although
combinatorial strategies using agents which target different protumorigenic
signaling pathway components have been shown to increase the clinical efficacy
of BRAF-I, novel strategies which utilize different antitumor mechanisms are
needed. An activating BRAF (V600E) kinase mutation occurs in approximately half of
melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032)
and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma
viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with
V600 BRAF mutant melanomas. However, acquired drug resistance invariably
develops after the initial treatment. Identification of acquired resistance
mechanisms may inform the development of new therapies that elicit long-term
responses of melanomas to BRAF inhibitors. Here we report that increased
expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired
resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly
upregulated in PLX4032-resistant melanoma cells because of the hyperactivation
of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway.
This upregulates AEBP1 expression and thus leads to the activation of NF-κB via
accelerating IκBa degradation. In addition, inhibition of the
PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant
phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is
validated in post-treatment tumors in patients with acquired resistance to BRAF
inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB
pathway whose activation contributes to acquired resistance to BRAF inhibition,
and suggest that this pathway, particularly AEBP1, may represent a novel
therapeutic target for treating BRAF inhibitor-resistant melanoma. RAF kinase inhibitors have substantial therapeutic effects in patients with
BRAF-mutant melanoma. However, only rarely do tumors regress completely, and the
therapeutic effects are often temporary. Several mechanisms of resistance to RAF
inhibitors have been proposed. The majority of these cause ERK signaling to
become insensitive to treatment with RAF inhibitors by increasing the amount of
RAF dimers in cells, whereas others bypass the dependence of the tumor on mutant
RAF. One motivation for studying mechanisms of drug resistance is that such
efforts may suggest new therapeutic targets or rational combination strategies
that delay or prevent the emergence of drug-resistant clones. Here, we review
the current model of RAF inhibitor resistance with a focus on the implications
of this model on ongoing laboratory and clinical efforts to develop more
effective therapeutic strategies for patients with BRAF-mutant tumors. Vemurafenib is a selective and potent small molecule inhibitor of the V600
mutant form of the BRAF protein used in the treatment of melanoma and colorectal
cancer. However, vemurafenib has less effect in BRAF mutant colorectal cancer
due to the resistance of tumor cell to vemurafenib. To verify whether or not
miR-145, a short RNA molecule of microRNA which has been supposed to be a tumor
suppressor, is involved in this process, we established vemurafenib-resistant
cell line colo205/V and found that the miR-145 expression was significantly
downregulated in colo205/V cells compared to normal colo205 cells. Moreover, the
overexpression of miR-145 could increase the sensitivity of colo205/V cells to
vemurafenib both in vitro and in vivo. In conclusion, miR-145 might be used as a
therapeutic target in the treatment of colorectal cancer patients with BRAF
V600E mutation. BACKGROUND: BRAF mutations occur in approximately 8% of all human cancers and
approach 50% in melanoma and papillary carcinoma of thyroid. These mutations
provide potentially valuable diagnostic, prognostic and treatment response
prediction markers. A sensitive, specific, low-cost assay to detect these
mutations is needed.
RESULTS: To detect BRAF V600E mutation in formalin-fixed, paraffin-embedded
(FFPE) tissue, we developed a method using Amplification Refractory Mutation
System (ARMS)-PCR. This method was designed to amplify three products in a
single reaction tube: a 200 bp common product serving as an amplification
control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt)
specific product. The sensitivity of this method was determined to be as low as
0.5% for the BRAF V600E allele in a wild-type background. This method was
successfully validated in 72 thyroid tumors. It detected V600E mutation in 22
out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of
12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular
variant of PTC showed BRAF V600E mutation. In addition, none of the 14
follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a
comparison method, direct dideoxy sequencing found only 27 out of 30 (90%)
mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has
higher sensitivity.
CONCLUSIONS: Our ARMS-PCR method provides a new tool for rapid detection of BRAF
V600E mutation. Our results indicate that ARMS-PCR is more sensitive than
automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE
tumor specimen. The strategy of this ARMS-PCR design may be adapted for early
detection of point mutations of a variety of biomarker genes. Personalized melanoma medicine has progressed from histopathologic features to
serum markers to molecular profiles. Since the identification of activating BRAF
mutations and subsequent development of drugs targeting the mutant BRAF protein,
oncologists now need to incorporate prognostic and predictive biomarkers into
treatment decisions for their melanoma patients. Examples include subgrouping
patients by genotype profiles for targeted therapy and the development of
serologic, immunohistochemical, and genotype profiles for the selection of
patients for immunotherapies. In this chapter, we provide an overview of the
current status of BRAF mutation testing, as well as promising serologic and
molecular profiles that will impact patient care. As further research helps
clarify the roles of these factors, the clinical outcomes of melanoma patients
promise to be greatly improved. The Braf(V600E) mutation has been detected in patients with metastatic melanoma,
colon, thyroid, and other cancers. Studies suggested that tumors with this
mutation are especially sensitive to BRAF inhibitors-hence the need to reliably
determine the BRAF status of tumor specimens. The present technologies used to
screen for this mutation fail to address the problems associated with
infiltrating stromal and immune cells bearing wild-type BRAF alleles and thus
may fail to detect the presence of mutant BRAF(V600E) tumors. We have developed
a rapid, inexpensive method of BRAF analysis that reduces the contamination of
wild-type BRAF sequences from tumor biopsies. The protocol involves a series of
PCR amplifications and restriction digestions that take advantage of unique
features of both wild-type and mutant BRAF RNA at codon 600. Using this
protocol, mutant BRAF can be detected in RNA from mixed populations with as few
as 0.1 % BRAF(V600E) mutant containing cells. (V600)BRAF mutation was identified as an ideal target for clinical therapy due
to its indispensable roles in supporting melanoma initiation and progression.
Despite the fact that BRAF inhibitors (BRAFi) can elicit anti-tumor responses in
the majority of treated patients and confer overall survival benefits, acquired
drug resistance is a formidable obstacle to long-term management of the disease.
Several aberrant events including RTK upregulation, NRAS mutation, mutant BRAF
amplification or alternative splicing, and MEK mutation have been reported as
acquired BRAFi resistance mechanisms. Clinially, detection of these resistance
mechanisms help understand drug response patterns and help guide combinatorial
therapeutic strategies. Therefore, quick and accurate diagnosis of the resistant
mechanisms in tumor biopsies has become an important starting point for
personalized therapy. In this chapter, we review the major acquired BRAFi
resistance mechanisms, highlight their therapeutic implications, and provide the
diagnostic methods from clinical samples. OBJECTIVE: To summarize the clinical development of dabrafenib and to highlight
the clinically relevant distinct characteristics of dabrafenib in contrast to
vemurafenib.
DATA SOURCE: An English-language literature search of MEDLINE/PubMed (1966-June
2013), using the keywords GSK2118436, dabrafenib, vemurafenib, selective BRAF
inhibitor, and advanced melanoma, was conducted. Data were also obtained from
package inserts, meeting abstracts, and clinical registries.
STUDY SELECTION AND DATA EXTRACTION: All relevant published articles on
dabrafenib and vemurafenib were reviewed. Clinical trial registries and meeting
abstracts were used for information about ongoing studies.
DATA SYNTHESIS: BRAF(V600E) mutation confers constitutive BRAK kinase activation
in melanoma cells, promoting tumor growth. This discovery led to the development
of BRAF kinase inhibitors like vemurafenib and dabrafenib. Dabrafenib has been
approved to treat patients with BRAF(V600E)-positive unresectable or metastatic
melanoma based on its clinical benefit demonstrated in a randomized phase III
study. It has also been shown to be safe and effective in patients with BRAF
mutant advanced melanoma involving the brain. Dabrafenib is well tolerated, with
the most common adverse effects being hyperkeratosis, headache, pyrexia, and
arthralgia. Currently, there is no evidence to suggest that one BRAF inhibitor
is superior to the other. With similar efficacy, therapy selection will likely
be influenced by differential tolerability and cost.
CONCLUSIONS: Dabrafenib joins vemurafenib to confirm the superior clinical
outcome of the BRAF inhibitors when compared with dacarbazine in patients with
BRAF(V600E)-positive advanced melanoma. Active research is ongoing to expand its
utility into the adjuvant setting and to circumvent rapid emergence of drug
resistance. Author information:
(1)1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard
Medical School; 2Department of Pathology, Massachusetts General Hospital Cancer
Center, Boston; 3Broad Institute of MIT and Harvard; 4Harvard-MIT Division of
Health Sciences and Technology, Massachusetts Institute of Technology (MIT),
Cambridge, Massachusetts; 5Department of Dermatology, University Hospital, West
German Cancer Center, University Duisburg-Essen, Essen; 6German Cancer
Consortium (DKTK); 7Department of Dermatology, Heidelberg University Hospital,
Heidelberg; 8Department of Dermatology and Allergy, Hannover Medical School,
Hannover; 9Department of Dermatology, University of Wuerzburg, Wuerzburg;
10Department of Dermatology, Venerology and Allergology, University of
Schleswig-Holstein Hospital, Kiel; 11Department of Dermatology and Allergology,
Ludwig-Maximilian University, Munich; 12Department of Dermatology, Venerology
and Allergy, Charité Universitätsmedizin Berlin, Humboldt University, Berlin;
13Department of Dermatology, University of Mainz, Mainz; 14University Medical
Center, University of Tübingen, Tübingen, Germany; 15Department of Genome
Sciences, University of Washington, Seattle, Washington; 16Department of
Dermatology, University Hospital Zurich, Zurich, Switzerland; and 17First
Department of Medicine, Medical School, University of Athens, Athens, Greece. Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which
target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases,
respectively, improves progression-free survival and response rates compared
with dabrafenib monotherapy. Mechanisms of clinical resistance to combined
RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and
whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant
tumors from five patients with acquired resistance to dabrafenib/trametinib. In
three of these patients, we identified additional mitogen-activated protein
kinase (MAPK) pathway alterations in the resistant tumor that were not detected
in the pretreatment tumor, including a novel activating mutation in MEK2
(MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in
vitro, but remained sensitive to inhibition of the downstream kinase
extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based
resistance identified in these patients suggests that alternative dosing of
current agents, more potent RAF/MEK inhibitors, and/or inhibition of the
downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma. BRAF inhibitors elicit rapid antitumor responses in the majority of patients
with BRAF(V600)-mutant melanoma, but acquired drug resistance is almost
universal. We sought to identify the core resistance pathways and the extent of
tumor heterogeneity during disease progression. We show that mitogen-activated
protein kinase reactivation mechanisms were detected among 70% of
disease-progressive tissues, with RAS mutations, mutant BRAF amplification, and
alternative splicing being most common. We also detected
PI3K-PTEN-AKT-upregulating genetic alterations among 22% of progressive
melanomas. Distinct molecular lesions in both core drug escape pathways were
commonly detected concurrently in the same tumor or among multiple tumors from
the same patient. Beyond harboring extensively heterogeneous resistance
mechanisms, melanoma regrowth emerging from BRAF inhibitor selection displayed
branched evolution marked by altered mutational spectra/signatures and increased
fitness. Thus, melanoma genomic heterogeneity contributes significantly to BRAF
inhibitor treatment failure, implying upfront, cotargeting of two core pathways
as an essential strategy for durable responses. BRAF inhibitors improve melanoma patient survival, but resistance invariably
develops. Here we report the discovery of a novel BRAF mutation that confers
resistance to PLX4032 employing whole-exome sequencing of drug-resistant
BRAF(V600K) melanoma cells. We further describe a new screening approach, a
genome-wide piggyBac mutagenesis screen that revealed clinically relevant
aberrations (N-terminal BRAF truncations and CRAF overexpression). The novel
BRAF mutation, a Leu505 to His substitution (BRAF(L505H) ), is the first
resistance-conferring second-site mutation identified in BRAF mutant cells. The
mutation replaces a small nonpolar amino acid at the BRAF-PLX4032 interface with
a larger polar residue. Moreover, we show that BRAF(L505H) , found in human
prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic
mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are
sensitive to novel, next-generation BRAF inhibitors, especially the
'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment
of melanoma patients with BRAF-mutations. BACKGROUND: Vemurafenib, an inhibitor of genetically activated BRAF, is now
commonly prescribed for metastatic melanoma harboring a BRAF mutation. Reports
on side effects have focused on cutaneous complications. We here present a case
of a severe pan-uveitis associated with vemurafenib use.
CASE PRESENTATION: A 63-year old female was treated with the BRAF inhibitor
vemurafenib for metastatic melanoma. After seven weeks of treatment, she
developed near-complete visual loss in the course of a few days, as a result of
severe uveitis. Vemurafenib had to be discontinued and systemic and topical
corticosteroids were initiated. The visual symptoms improved slowly, however the
cerebral metastases progressed and the patient died from her disease.
CONCLUSION: Treatment with vemurafenib has become an important component of
standard clinical care for patients with metastatic melanoma. In addition, it is
one of the best examples of genotype-directed therapy. This case illustrates
that vemurafenib-induced uveitis can develop fast and be slow to resolve.
Awareness of this potentially severe side effect is of major importance to
oncologists and aggressive treatment should be considered. Melanoma is the most aggressive form of skin cancer. The treatment of patients
with advanced melanoma is rapidly evolving due to an improved understanding of
molecular drivers of this disease. Somatic mutations in BRAF are the most common
genetic alteration found in these tumors. Recently, two different
mutant-selective small molecule inhibitors of BRAF, vemurafenib and dabrafenib,
have gained regulatory approval based on positive results in randomized phase
III trials. While the development of these agents represents a landmark in the
treatment of melanoma, the benefit of these agents is limited by the frequent
and rapid onset of resistance. The identification of several molecular
mechanisms of resistance to BRAF inhibitors is rapidly leading to the clinical
testing of combinatorial strategies to improve the clinical benefit of these
agents. These mechanisms, and the lessons learned from the initial testing of
the BRAF inhibitors, provide multiple insights that may facilitate the
development of targeted therapies against other oncogenic mutations in melanoma,
as well as in other cancers. BACKGROUND & AIM: Brain metastases are frequent in patients with metastatic
melanoma, indicating poor prognosis. We investigated the BRAF kinase inhibitor
vemurafenib in patients with advanced melanoma with symptomatic brain
metastases.
METHODS: This open-label trial assessed vemurafenib (960mg twice a day) in
patients with BRAF(V600) mutation-positive metastatic melanoma with
non-resectable, previously treated brain metastases. The primary end-point was
safety. Secondary end-points included best overall response rate, and
progression-free and overall survival.
RESULTS: Twenty-four patients received vemurafenib for a median treatment
duration of 3.8 (0.1-11.3) months. The majority of discontinuations were due to
disease progression (n=22). Twenty-three of 24 patients reported at least one
adverse event (AE). Grade 3 AEs were reported in four (17%; 95% confidence
interval [CI], 4.7-37.4%) patients and included cutaneous squamous cell
carcinoma in four patients. Median progression-free survival was 3.9 (95% CI,
3.0-5.5) months, and median survival was 5.3 (95% CI, 3.9-6.6) months. An
overall partial response (PR) at both intracranial and extracranial sites was
achieved in 10 of 24 (42%; 95% CI, 22.1-63.4) evaluable patients, with stable
disease in nine (38%; 95% CI, 18.8-59.4) patients. Of 19 patients with
measurable intracranial disease, seven (37%) achieved >30% intracranial tumour
regression, and three (16%; 95% CI, 3.4-39.6%) achieved a confirmed PR. Other
signs of improvement included reduced need for corticosteroids and enhanced
performance status.
CONCLUSIONS: Vemurafenib can be safely used in patients with advanced
symptomatic melanoma that has metastasised to the brain and can result in
meaningful tumour regression. Different genetic aberrations of BRAF have been reported in various
maligcies. BRAF is member of the RAS/RAF/MEK/ERK pathway and constitutive
activity of this pathway can lead to increased cellular growth, invasion, and
metastasis. The most common activating BRAF mutation in colorectal cancer is the
V600E mutation, which is present in 5-15% of all tumors, and up to 80% of tumors
with high microsatellite instability (MSI) harbor this mutation. BRAF mutation
is associated with proximal location, higher age, female gender, MSI-H, high
grade, and mucinous histology, and is a marker of poor prognosis in colorectal
cancer. The role of BRAF mutation as a predictive marker in respect of EGFR
targeted treatments is controversial. BRAF V600 selective inhibitors have been
approved for the treatment of V600 mutation positive metastatic melanoma, but
the response rates in colorectal cancer are poor. This might be due to innate
resistance mechanisms of colorectal cancers against the treatment solely
targeting BRAF. To overcome resistance the combination of treatments,
simultaneous inhibition of BRAF and MEK or PI3K/mTOR, might emerge as a
successful therapeutic concept. BRAF mutations have emerged as an important predictive biomarker for
metastasized melanoma. Other types of cancer may also benefit from BRAF
mutation-targeted therapies. In biliary tract cancer, reported BRAF mutation
rates are highly controversial, ranging from 0 to 33% in adenocarcinoma of the
gallbladder and 0 to 22% in cholangiocarcinoma. We here analyzed tissue
microarrays of a large cohort of biliary tract cancer (n=377) including 159
intrahepatic cholangiocarcinomas, 149 extrahepatic cholangiocarcinomas, and 69
adenocarcinomas of the gallbladder for BRAF V600E mutation using a highly
sensitive immunohistochemical screening approach implementing the BRAF V600E
protein-specific antibody VE1. All VE1-positive cases as well as 42 VE1-negative
cases were additionally analyzed by Sanger sequencing. In total, only 5
VE1-positive cases were detected (5/377; 1%). BRAF V600E mutation was confirmed
by direct sequencing in all cases. All 5 mutated cases were intrahepatic
cholangiocarcinomas (5/159; 3%). None of the extrahepatic cholangiocarcinomas
and adenocarcinomas of the gallbladder were VE1 positive. Apart from the subtype
restriction of BRAF V600E mutation to intrahepatic cholangiocarcinoma and a
female predomice (4 female, 1 male), no significant correlation with
clinicopathological data and patient outcome was detected. In conclusion, we
demonstrate that BRAF V600E mutation is a rare event in biliary tract cancer,
accounting for only 1% of all subtypes, and is restricted to intrahepatic
cholangiocarcinoma. In addition, we demonstrate that VE1 immunohistochemistry is
a feasible approach to routinely screen for BRAF V600E mutation in biliary tract
cancer patients, thereby facilitating the detection of rare patients who may
benefit from BRAF mutation-targeted therapies. Activating BRAF mutations, leading to constitutive activation of the MAPK
signaling pathway, are common in a variety of human cancers. Several small
molecule BRAF inhibitors have been developed during the last years and shown
promising results in clinical trials, especially for metastatic melanoma, while
they have been less effective in colon cancer. Two inhibitors, vemurafenib and
dabrafenib, have been approved for treatment of melanoma. Unfortunately, in most
patients who initially respond the tumors eventually develop acquired resistance
to the BRAF inhibitors. So far, a number of resistance mechanisms have been
identified, including secondary NRAS mutations and BRAF alternative splicing,
leading to reactivation of the MAPK pathway. Other alterations, both upstream
and downstream of BRAF can have the same effect, and activation of alternative
pathways can also play a role in resistance to BRAF inhibitors. In addition,
intra-tumor heterogeneity with the presence of clones of tumor cells lacking
BRAF mutations needs to be considered, since wildtype BRAF can be activated by
inhibitors designed to target mutated BRAF. Combination of the BRAF inhibitor
dabrafenib with the MEK inhibitor trametinib has significantly prolonged
progression free survival compared to dabrafenib alone in metastatic melanoma.
Combination treatments of BRAF inhibitors with other agents may not only
circumvent or delay resistance, but may also lead to fewer side effects, such as
development of secondary squamous tumors. Several clinical trials are underway
for many different BRAF mutation positive cancers with BRAF inhibitors alone or
in combination with other small molecule inhibitors, immunotherapies or
conventional chemotherapy. |
Is it possible to determine the proteome of a formalin fixed and paraffin embedded (FFPE) tissue? | Yes, advances in sample preparation has enabled the proteomic analysis of formalin-fixed and paraffin-embedded tissues. | Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially
valuable resource for retrospective biomarker discovery studies, and recent work
indicates the feasibility of using shotgun proteomics to characterize FFPE
tissue proteins. A critical question in the field is whether proteomes
characterized in FFPE specimens are equivalent to proteomes in corresponding
fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of
frozen and FFPE specimens prepared from the same colon adenoma tissues.
Following deparaffinization, rehydration, and tryptic digestion under mild
conditions, FFPE specimens corresponding to 200 microg of protein yielded
approximately 400 confident protein identifications in a one-dimensional reverse
phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The
major difference between frozen and FFPE proteomes was a decrease in the
proportions of lysine C-terminal to arginine C-terminal peptides observed, but
these differences had little effect on the proteins identified. No covalent
peptide modifications attributable to formaldehyde chemistry were detected by
analyses of the MS/MS datasets, which suggests that undetected, cross-linked
peptides comprise the major class of modifications in FFPE tissues. Fixation of
tissue for up to 2 days in neutral buffered formalin did not adversely impact
protein identifications. Analysis of archival colon adenoma FFPE specimens
indicated equivalent numbers of MS/MS spectral counts and protein group
identifications from specimens stored for 1, 3, 5, and 10 years. Combination of
peptide isoelectric focusing-based separation with reverse phase LC-MS/MS
identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302
protein groups from FFPE tissue with at least two distinct peptide
identifications per protein. Analysis of the combined frozen and FFPE data
showed a 92% overlap in the protein groups identified. Comparison of gene
ontology categories of identified proteins revealed no bias in protein
identification based on subcellular localization. Although the status of
posttranslational modifications was not examined in this study, archival samples
displayed a modest increase in methionine oxidation, from approximately 17%
after one year of storage to approximately 25% after 10 years. These data
demonstrate the equivalence of proteome inventories obtained from FFPE and
frozen tissue specimens and provide support for retrospective proteomic analysis
of FFPE tissues for biomarker discovery. Global mass spectrometry (MS) profiling and spectral count quantitation are used
to identify unique or differentially expressed proteins and can help identify
potential biomarkers. MS has rarely been conducted in retrospective studies,
because historically, available samples for protein analyses were limited to
formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable
methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic
analysis of these samples has been confounded by formalin-induced protein
cross-linking. The performance of extracted proteins in a liquid chromatography
tandem MS format from FFPE samples and extracts from whole and laser capture
microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)-embedded
matched control rat liver samples were compared. Extracts from FFPE and
frozen/OCT-embedded livers from atorvastatin-treated rats were further compared
to assess the performance of FFPE samples in identifying atorvastatin-regulated
proteins. Comparable molecular mass representation was found in extracts from
FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less
for the FFPE sample. The numbers of shared proteins identified indicated that
robust proteomic representation from FFPE tissue and LCM did not negatively
affect the number of identified proteins from either OCT-frozen or FFPE samples.
Subcellular representation in FFPE samples was similar to OCT-frozen, with
predomitly cytoplasmic proteins identified. Biologically relevant protein
changes were detected in atorvastatin-treated FFPE liver samples, and selected
atorvastatin-related proteins identified by MS were confirmed by Western blot
analysis. These findings demonstrate that formalin fixation, paraffin
processing, and LCM do not negatively impact protein quality and quantity as
determined by MS and that FFPE samples are amenable to global proteomic
analysis. This unit describes a method of isolating of proteins from formalin-fixed and
paraffin-embedded (FFPE) tissue for mass spectrometry analysis. Heat-induced
antigen retrieval is the basis of the protein extraction strategy presented in
this protocol. This protocol may be used to identify nuclear, cytosolic, and
membrane proteins from FFPE tissues extracted from tissue blocks or slides. This unit provides a robust, reliable, and easy-to-use kit-based method for
extraction of intact, non-degraded proteins from formalin-fixed,
paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid
chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are
extracted from unstained sections of FFPE rat liver tissue. After a simple
cleanup step using organic extraction, the sample is transferred into a buffer
optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS
is used to identify the proteins that gave rise to the tryptic peptides.
Comparing formalin-fixed and frozen tissues, good correlation is observed in the
mass spectrometric pattern attributable to the tryptic peptides and number of
identified proteins. Since FFPE tissues are generally available in clinical
practice, this method can be used to analyze biomarkers in different
pathological situations (e.g., healthy vs. diseased). The method can also be
used for protein extraction from fresh-frozen tissue. Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded
(FFPE), in which form they are preserved for decades. It has only recently been
shown that proteins in FFPE tissues can be identified by mass spectrometry-based
proteomics but analysis of post-translational modifications is thought to be
difficult or impossible. The filter aided sample preparation (FASP) method can
analyze proteomic samples solubilized in high concentrations of SDS and we use
this feature to develop a simple protocol for FFPE analysis. Combination with
simple pipet-tip based peptide fractionation identified about 5000 mouse liver
proteins in 24 h measurement time-the same as in fresh tissue. Results from the
FFPE-FASP procedure do not indicate any discernible changes due to storage time,
hematoxylin staining or laser capture microdissection. We compared fresh against
FFPE tissue using the SILAC mouse and found no significant qualitative or
quantitative differences between these samples either at the protein or the
peptide level. Application of our FFPE-FASP protocol to phosphorylation and
N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation
sites. Analysis of FFPE tissue of the SILAC mouse revealed that these
post-translational modifications were quantitatively preserved. Thus, FFPE
biobank material can be analyzed by quantitative proteomics at the level of
proteins and post-translational modifications. Imaging mass spectrometry (IMS) is a powerful technology for mapping
distributions of biological molecules like proteins and peptides within tissue
sections. It is therefore potentially extremely useful for the analysis of
pathological conditions such as neoplastic diseases. The use of IMS is typically
limited to fresh frozen tissue specimens. However, there is a high interest in
the possibility of being able to analyze the tissue proteome of formalin-fixed
paraffin-embedded (FFPE) specimens that have been stored together with the
clinicopathological information of patients in huge archives over many decades.
We have therefore developed an antigen-retrieval protocol using a high
temperature citric acid buffer to allow partial reversal of FFPE protein
cross-linking. Coupled with automated deposition of trypsin and matrix, our
method allows the generation of meaningful peptide ion distribution images. In
situ peptide fragmentation provided identification of high abundance proteins
such as Actin and Collagen. Furthermore, downstream application of three
different HPLC-MS strategies allowed identification of a maximum of 106
proteins, 67 of which were mass correlated to ions from IMS analysis of archived
FFPE ovarian tissue. The CAAR method presented here complements previously
described antigen-retrieval protocols and is an important step in being able to
fully analyze the proteome of archived FFPE tissue. Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as
an alternative to fresh/frozen tissue for retrospective protein biomarker
discovery. However, during the fixation process, proteins undergo degradation
and cross-linking, making conventional protein analysis technologies
problematic. In this study, we have compared several extraction and separation
methods for the analysis of proteins in FFPE tissues. Incubation of tissue
sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH
8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture
of protease inhibitors) resulted in improved protein recovery. Protein
separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective
approach to identify proteins, based on the number of peptides reliably
identified. Interestingly, a number of peptides were identified in regions of
the 1DE not corresponding to their native molecular weights. This is an
indication of the formation of protein-protein complexes by cross-linking, and
of protein fragmentation due to prolonged sample storage. This study will
facilitate the development of future proteomic analysis of FFPE tissue and
provide a tool for the validation in archival samples of biomarkers of exposure,
prognosis and disease. Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a
valuable resource for retrospective biomarker discovery. However, proteomic
exploration of archival tissue is impeded by extensive formalin-induced covalent
cross-linking. Robust methodology enabling proteomic profiling of archival
resources is urgently needed. Recent work is beginning to support the
feasibility of biomarker discovery in archival tissues, but further developments
in extraction methods which are compatible with quantitative approaches are
urgently needed. We report a cost-effective extraction methodology permitting
quantitative proteomic analyses of small amounts of FFPE tissue for biomarker
investigation. This surfactant/heat-based approach results in effective and
reproducible protein extraction in FFPE tissue blocks. In combination with a
liquid chromatography-mass spectrometry-based label-free quantitative proteomics
methodology, the protocol enables the robust representative and quantitative
analyses of the archival proteome. Preliminary validation studies in renal
cancer tissues have identified typically 250-300 proteins per 500 ng of tissue
with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue
blocks and preservation of tumor/normal differential expression patterns (205
proteins, r = 0.682; p < 10(-15)). The initial methodology presented here
provides a quantitative approach for assessing the potential suitability of the
vast FFPE tissue archives as an alternate resource for biomarker discovery and
will allow exploration of methods to increase depth of coverage and investigate
the impact of preanalytical factors. Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated
diagnostic records represent an invaluable source of proteomic information on
diseases where the patient outcomes are already known. Over the last few years,
advances in methodology have made it possible to recover peptides from FFPE
tissues that yield a reasonable representation of the proteins recovered from
identical fresh or frozen specimens. These new methods, based largely upon
heat-induced antigen retrieval techniques borrowed from immunohistochemistry,
have developed sufficiently to allow at least a qualitative analysis of the
proteome of FFPE archival tissues. This chapter describes the approaches for
performing proteomic analysis on FFPE tissues by liquid chromatography and mass
spectrometry. Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related
death, largely due to metastatic disease. To better understand PDAC metastatic
spread and identify novel therapeutic targets, we analysed the proteome of
primary tumours and matched lymph node (LN) metastases. As frozen specimens of
metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded
(FFPE) tissues. This poses technical challenges because of the cross-linkages
induced by fixation. Using laser capture microdissection (PALM system), we
isolated maligt epithelia from seven FFPE primary PDAC tumours and matched LN
metastases. Following dissection, samples were analysed in duplicate using
Multidimensional Protein Identification Technology (MudPIT); this resulted in
the identification of 1504 proteins, 854 of which were common to all samples
analysed. Comparison of the obtained proteins with data from previous proteomics
studies on pancreatic tissue, pancreatic juice, serum, and urine resulted in a
less than 30% overlap, indicating that our study has substantially expanded the
current database of proteins expressed in this maligcy. Statistical analysis
further showed that 115/854 proteins (13.5%) were significantly differentially
expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly
significant g-values were confirmed to be significantly differentially expressed
(S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) in a larger series of 55 cases of
matched primary PDAC and LN metastases using immunohistochemistry. Thus, laser
capture microdissection of FFPE tissue coupled with downstream proteomic
analysis is a valid approach for the investigation of metastatic PDAC. This is
the first study to establish and compare the protein composition of primary PDAC
and matched LN metastases, and has resulted in the identification of several
potential epithelial-specific therapeutic targets, including 14-3-3 sigma and
S100P. Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not
been widely used in neuroproteomic studies because many proteins are presumed to
be degraded during tissue preservation. Recent improvements in proteomics
technologies, from the 2D gel analysis of intact proteins to the "shotgun"
quantification of peptides and the use of isobaric tags for absolute and
relative quantification (iTRAQ) method, have made the analysis of FFPE tissues
possible. In recent years, iTRAQ has been one of the main methods of choice for
high throughput quantitative proteomics analysis, which enables simultaneous
comparison of up to eight samples in one experiment. Our objective was to assess
the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous
tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced
proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced
proteomic changes in FFPE tissues were positively correlated with those found in
the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent
validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal
cords led us to evaluate other proteasome and IP-specific proteins. We
discovered that many IP-specific (as opposed to constitutive) proteasomal
proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation
also occurred in the spinal cords of an independent mouse EAE model in a
disability score-dependent manner. Therefore, we conclude that it is feasible to
generate useful information from iTRAQ-based neuroproteomics analysis of
archived FFPE tissues for studying neurological disease tissues. Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially
valuable resource for both prospective and retrospective biomarker discovery.
Unlocking the proteomic profile of clinicopathological FFPE tissues is a
critically essential step for annotating clinical findings and predicting
biomarkers for ultimate disease prognosis and therapeutic follow-up. Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a
valuable resource for the retrospective study of disease progression and
response to therapy. However, the proteomic analysis of FFPE tissues has been
hampered by formaldehyde-induced protein modifications, which reduce protein
extraction efficiency and may lead to protein misidentification. Here, we
demonstrate the use of heat augmented with high hydrostatic pressure (40,000
psi) as a novel method for the recovery of intact proteins from FFPE mouse
liver. When FFPE mouse liver was extracted using heat and elevated pressure,
there was a 4-fold increase in protein extraction efficiency, a 3-fold increase
in the extraction of intact proteins, and up to a 30-fold increase in the number
of nonredundant proteins identified by mass spectrometry, compared to matched
tissue extracted with heat alone. More importantly, the number of nonredundant
proteins identified in the FFPE tissue was nearly identical to that of matched
fresh-frozen tissue. |
What is known about type D personality trait in cancer patients? | Reported prevalence rates of type D personality ranges from 19% to 22% in patients with cancer. In patients with cancer, Type D personality is associated with poor quality of life and mental health. Cancer patients with a Type D personality as compared with non-Type D patients perceive that they receive less information, report less satisfaction with the amount of received information, believe that their illness has significantly more serious consequences, will last significantly longer, and experience significantly more symptoms that they attribute to their illness. Also, they are more concerned about their illness, and their disease more often influences them emotionally. Also, Type D cancer patients are at an increased risk for comorbidity burden and increased health care utilization. | OBJECTIVE: We aimed to investigate the prevalence of Type D personality (the
conjoint effects of negative affectivity and social inhibition) among melanoma
survivors and to obtain insight into its effects on health status, impact of
cancer and health care utilisation.
METHODS: We selected all patients diagnosed with melanoma between 1998 and 2007
from three large regional hospitals in the Netherlands. In total, 699 survivors,
alive in January 2008, received a questionnaire including Type D personality
scale (DS14), impact of cancer questionnaire (IOC) and SF-36 and 80% responded
(n=562).
RESULTS: Twenty-two percent of survivors (n=125) were classified as Type D. They
reported a clinically and statistically significant worse general health (57.8
versus 75.6), social functioning (73.1 versus 88.7), mental health (61.7 versus
80.6), more emotional role limitations (67.8 versus 89.4) and less vitality
(54.5 versus 72.8) than non-Type D patients. Additionally, they reported a
statistically and clinically relevant higher impact of cancer on body changes,
negative self-evaluation, negative outlook on life, life interferences and
health worry. Furthermore, they were more worried about the influence of the sun
on their skin and acted accordingly. No differences were found in health care
utilisation.
CONCLUSIONS: Type D personality has a distinct negative impact on health status
in melanoma survivors and is an important factor to screen for in clinical
practice. Giving special attention to these patients is important while they are
more likely to experience a strong impact of cancer which cannot be explained by
socio-demographical or clinical characteristics. PURPOSE/OBJECTIVES: To explore the association between quality of life (QOL) and
type D personality, which is characterized by the traits of negative affectivity
and social inhibition, and to further identify impacts of these traits after
controlling for biophysical and psychological factors in colorectal cancer
survivors.
DESIGN: Cross-sectional and correlational.
SETTING: Oncology and surgical outpatient clinics of a medical center in Taiwan.
SAMPLE: 124 patients diagnosed with colorectal cancer who had completed active
treatment.
METHODS: Data were collected using a set of structured questionnaires to explore
type D personality, biophysical and psychological factors, and QOL. Their
associations were verified with Mann-Whitney U test and Spearman's rho
correlation. Significant factors associated with QOL were identified with
generalized estimating equations.
MAIN RESEARCH VARIABLES: Type D personality and QOL.
FINDINGS: Patients with type D personality experienced higher physical and
psychological distress than those with non-type D personality. Social inhibition
remained an important factor leading to impairment in the mental component of
QOL after controlling for other associated factors. Negative affectivity was
associated with fatigue intensity and interference of fatigue with life
activities.
CONCLUSIONS: Personality trait was found to be an important factor associated
with QOL. The trait of social inhibition was a significant factor influencing
mental aspects of QOL, whereas negative affectivity was associated with fatigue.
IMPLICATIONS FOR NURSING: Assessing patients' personality, including negative
affectivity and social inhibition, could help nurses to develop supportive
groups or social networks for these patients and thereby improve QOL for cancer
survivors. BACKGROUND: This study assessed the association between Type D personality (the
conjoint effect of negative affectivity and social inhibition) and quality of
life (QoL) and mental health of cancer survivors up to 10 years post-diagnosis.
METHODS: All currently alive individuals diagnosed with endometrial or
colorectal cancer between 1998 and 2007, or with lymphoma or multiple myeloma
between 1999 and 2008 as registered in the Eindhoven Cancer Registry received a
questionnaire on Type D personality (DS14), QoL (SF-36 or EORTC-QLQ-C30) and
mental health (HADS).
RESULTS: Of the 3080 survivors who responded (69%), 572 (19%) had a Type D
personality. Type D survivors had clinically meaningful lower levels of general
health, social functioning, role-function emotional, mental health and vitality
compared to non-Type D's (SF-36: all P's<0.001). They also reported clinically
meaningful worse emotional and social functioning, global health status/QoL, and
more fatigue (EORTC-QLQ-C30: all P's<0.001). This was also confirmed by
multivariate logistic regression analyses showing that cancer survivors with a
Type D personality were more likely to experience a decreased QoL on all SF-36
and EORTC-QLQ-C30 scales (all ORs ranging between 1.88 and 5.56). The proportion
of survivors reporting an impaired QoL was higher among Type D (35-64%) than
non-Type D's (20-36%). Finally, Type D's were more likely to be depressed (44%
vs. 13%; P<0.0001) or anxious (51% vs. 14%; P<0.0001).
CONCLUSIONS: Cancer survivors with a Type D personality are at increased risk of
impaired QoL and mental health problems that cannot be explained by
socio-demographic or clinical characteristics. OBJECTIVE: Cancer survivors often report comorbid diseases, but there are
individual differences in risk. Type D personality is a general propensity to
psychological distress that is related to poor cardiovascular outcomes. In this
study, we examined whether type D was also related to comorbidity burden and
health care utilization among cancer survivors.
METHODS: Individuals diagnosed with endometrial cancer or colorectal cancer
between 1998 and 2007, or with lymphoma or multiple myeloma between 1999 and
2008 as registered in the Eindhoven Cancer Registry, received the
Self-Administered Comorbidity Questionnaire, questions on health care
utilization and the Type D personality scale; 69% (n=3080) responded.
RESULTS: Nineteen percent of survivors had a type D personality. Over a 12-month
period, type D survivors significantly more often reported osteoarthritis, back
pain, and depression than non-type D survivors. Also, type D survivors more
often reported to feel bothered by high blood pressure, osteoarthritis, heart
disease, depression, diabetes and lung disease during daily activities. Type D
survivors more often visited their general practitioner than non-type D
survivors (P<.001), also in relation to cancer (0 visits: 54% vs. 60%; 1-5: 28%
vs. 22%; >5: 9% vs. 5%; P<.001), as well as their specialist (0 visits: 6% vs.
7%; 1-5 visits: 59% vs. 64%; >5 visits: 30% vs. 23%; P<.01).
CONCLUSION: Type D personality is a vulnerability factor that may help to
identify subgroups of cancer survivors who are at an increased risk for
comorbidity burden and increased health care utilization. OBJECTIVE: To examine the association between Type D personality and illness
perceptions among colorectal cancer survivors 1-10years post-diagnosis.
METHODS: Data from two population-based surveys on colorectal cancer survivors
was used. Patients diagnosed between 1998 and 2009, as registered in the
Eindhoven Cancer Registry, received a questionnaire on Type D personality (DS14)
and illness perceptions (B-IPQ); 81% (n=3977) responded.
RESULTS: 750 (19%) patients had a Type D personality. They believe their illness
has significantly more serious consequences, will last significantly longer, and
experience significantly more symptoms that they attribute to their illness.
Also, they are more concerned about their illness, and their disease more often
influences them emotionally. Differences regarding 'consequences', 'concern' and
'emotional response' were also clinically relevant. The majority of patients
stated that the cause of their disease was unknown (23.3%), hereditary (20.3%),
lifestyle (15.1%), psychological distress (11.9%) or other (11.6%). Significant
differences in perceptions on cause of disease between Type Ds and non-Type Ds
were found for psychological distress (16.2 vs. 10.9%; p<0.01), randomness (1.7
vs. 5.3%; p<0.01) and unknown (18.8 vs. 24.4%; p<0.01). Multivariate analyses
showed that Type D was negatively associated with 'coherence' and positively
with 'consequences', 'timeline', 'identity', 'concern', and 'emotional
representation'.
CONCLUSIONS: These results elucidate the associations between personality and
illness perceptions, demonstrating their close interrelatedness. Our study may
be helpful in further developing theoretical models regarding giving meaning to
illness and the illness perceptions that the illness elicits. Future studies
should investigate whether interventions can positively impact illness
perceptions of Type D cancer patients. |
List available tools for genomic visualisation in comparative genomics | Insyght, Genomicus and Sockeye. | Comparative genomics techniques are used in bioinformatics analyses to identify
the structural and functional properties of DNA sequences. As the amount of
available sequence data steadily increases, the ability to perform large-scale
comparative analyses has become increasingly relevant. In addition, the growing
complexity of genomic feature annotation means that new approaches to genomic
visualization need to be explored. We have developed a Java-based application
called Sockeye that uses three-dimensional (3D) graphics technology to
facilitate the visualization of annotation and conservation across multiple
sequences. This software uses the Ensembl database project to import sequence
and annotation information from several eukaryotic species. A user can
additionally import their own custom sequence and annotation data. Individual
annotation objects are displayed in Sockeye by using custom 3D models.
Ensembl-derived and imported sequences can be analyzed by using a suite of
multiple and pair-wise alignment algorithms. The results of these comparative
analyses are also displayed in the 3D environment of Sockeye. By using the
Java3D API to visualize genomic data in a 3D environment, we are able to
compactly display cross-sequence comparisons. This provides the user with a
novel platform for visualizing and comparing genomic feature organization. High-throughput techniques have considerably increased the potential of
comparative genomics whilst simultaneously posing many new challenges. One of
those challenges involves efficiently mining the large amount of data produced
and exploring the landscape of both conserved and idiosyncratic genomic regions
across multiple genomes. Domains of application of these analyses are diverse:
identification of evolutionary events, inference of gene functions, detection of
niche-specific genes or phylogenetic profiling. Insyght is a comparative genomic
visualization tool that combines three complementary displays: (i) a table for
thoroughly browsing amongst homologues, (ii) a comparator of orthologue
functional annotations and (iii) a genomic organization view designed to improve
the legibility of rearrangements and distinctive loci. The latter display
combines symbolic and proportional graphical paradigms. Synchronized navigation
across multiple species and interoperability between the views are core features
of Insyght. A gene filter mechanism is provided that helps the user to build a
biologically relevant gene set according to multiple criteria such as
presence/absence of homologues and/or various annotations. We illustrate the use
of Insyght with scenarios. Currently, only Bacteria and Archaea are supported. A
public instance is available at http://genome.jouy.inra.fr/Insyght. The tool is
freely downloadable for private data set analysis. |
Which species may be used for the biotechnological production of itaconic acid? | In 1955, the production of itaconic acid was firstly described for Ustilago maydis. Some Aspergillus species, like A. itaconicus and A. terreus, show the ability to synthesize this organic acid and A. terreus can secrete significant amounts to the media. Itaconic acid is mainly supplied by biotechnological processes with the fungus Aspergillus terreus. Cloning of the cadA gene into the citric acid producing fungus A. niger showed that it is possible to produce itaconic acid also in a different host organism. | The continuous itaconic acid production from sucrose with Aspergillus terreus
TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in
column bioreactors using statistical experimental design and empirical
modelling. The highest itaconic acid product concentration calculated on the
basis of the obtained model was 15.8 g l-1 in the investigated experimental
area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and
residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3
mycelium itaconic acid production was stable for at least 4.5 months in
continuous column bioreactors. In comparison, using glucose as substrate and
immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was
obtained with higher product concentration. The omission of copper sulphate from
the production medium gave the highest itaconic acid product concentration (26 g
l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate. A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated
from horticulture waste. Market refuse, apple and baa, were explored as novel
substrates for itaconic acid production with yields of 20+/-2.0 and 20.0+/-1.0 g
l(-1), respectively. Itaconic acid yields of 28.5+/-2.2 and 31.0+/-1.7 g l(-1)
were obtained with acid and alpha-amylase hydrolyzed corn starch. The efficiency
of itaconic acid production by this wild type strain was improved by
ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid
yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two
mutants were capable of producing twice the yield of itaconic acid as the parent
strain. Sago starch was hydrolyzed using either chemical agents, or enzymes at various
pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase
exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas
hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium
was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep
liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of
hydrolysis and medium composition were applied to itaconic acid production in a
3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of
0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of
itaconic acid was recovered with a purity of 97.2%. This result showed that sago
starch could be converted to a value-added product with only a simple
pretreatment. Fermentation products of Aspergillus terreus ATCC 20542 (a parent strain for
lovastatin production) were collected, and the coexistence of itaconic acid (IA)
with lovastatin was confirmed in this study. Using a lactose-based medium (LBM),
lovastatin production was 873 mg/l on day 10, but IA production was only 22-28
mg/l during the cultures. When lactose in LBM was simply replaced with glucose,
IA production was markedly enhanced by 20-fold (491 mg/l on day 5), which showed
a growth-associated pattern. The findings indicated that the carbon source used
(glucose or lactose) controlled the biosynthetic pathway. The net yield of
lovastatin production when using lactose was calculated to be 25.1 mg/g
(5.1-fold) in comparison with when using glucose in the cultures. Furthermore,
lovastatin production was further increased by 9.2% when IA (0.5 g/l) was added
to LBM. When IA was added at 5 g/l, the fermentation broth turned dark-brown,
and lovastatin production was reduced by 18.0%. Hence, these two metabolites (IA
and lovastatin) produced by the fungus might be related. Aspergillus terreus is successfully used for industrial production of itaconic
acid. The acid is formed from cis-aconitate, an intermediate of the
tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase.
It could be assumed that strong anaplerotic reactions that replenish the pool of
the TCA cycle intermediates would enhance the synthesis and excretion rate of
itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated
metabolic flux through glycolysis has been described that acted as a strong
anaplerotic reaction. Deregulated glycolytic flux was caused by
posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted
in formation of a highly active, citrate inhibition-resistant shorter form of
the enzyme. In order to avoid complex posttranslational modification, the native
A. niger pfkA gene has been modified to encode for an active shorter PFK1
fragment. By the insertion of the modified A. niger pfkA genes into the A.
terreus strain, increased specific productivities of itaconic acid and final
yields were documented by transformants in respect to the parental strain. On
the other hand, growth rate of all transformants remained suppressed which is
due to the low initial pH value of the medium, one of the prerequisites for the
accumulation of itaconic acid by A. terreus mycelium. Several Aspergillus species are well-known for the production of a variety of
organic acids. In this study, a cloned based transcriptomics approach was used
to identify genes crucial in the biosynthesis pathway for one of these acids,
itaconic acid. From a number of different Aspergillus terreus controlled batch
fermentations, those cultures with the largest difference in itaconic acid titer
and productivity were selected for mRNA isolation. cDNAs derived from these mRNA
samples were used for subsequent hybridization of glass slide arrays based on a
collection of 5000 cDNA clones. A selection of 13 cDNA clones resulting in the
strongest (>10-fold) differential hybridization signals were identified and
subsequently the inserts of these clones were sequenced. Sequence analysis
revealed the presence of in total five different gene inserts among the
sequenced clones. From one of these sequences, encoding a gene of the MmgE-PrpD
family, the full length coding region was shown to encode one of the crucial
itaconic acid pathway enzymes cis-aconitate decarboxylase, by heterologous
expression in Escherichia coli. Expression of this gene in Aspergillus niger,
which is a natural citric acid producer, resulted in itaconate production.
Genome analysis suggests that in A. terreus the cis-aconitate decarboxylase gene
is part of an itaconate acid related gene cluster including genes encoding two
pathway specific transporters and a Zinc finger protein. Interestingly, this
cluster is directly linked to the large lovastatin gene cluster. BACKGROUND: In the last years, the biotechnological production of platform
chemicals for fuel components has become a major focus of interest. Although
ligno-cellulosic material is considered as suitable feedstock, the almost
inevitable pretreatment of this recalcitrant material may interfere with the
subsequent fermentation steps. In this study, the fungus Ustilago maydis was
used to produce itaconic acid as platform chemical for the synthesis of
potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have
investigated how pretreatment of ligno-cellulosic biomass precisely influences
the subsequent fermentation by U. maydis. Thus, this current study aims to first
characterize U. maydis in shake flasks and then to evaluate the influence of
three exemplary pretreatment methods on the cultivation and itaconic acid
production of this fungus. Cellulose enzymatically hydrolysed in seawater and
salt-assisted organic-acid catalysed cellulose were investigated as substrates.
Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as
substrate.
RESULTS: U. maydis was characterized on shake flask level regarding its itaconic
acid production on glucose. Nitrogen limitation was shown to be a crucial
condition for the production of itaconic acid. For itaconic acid concentrations
above 25 g/L, a significant product inhibition was observed. Performing
experiments that simulated influences of possible pretreatment methods, U.
maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well
as of 0.1 M oxalic acid. The production of itaconic acid was achieved on
pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction
of pretreated beech wood.
CONCLUSION: The fungus U. maydis is a promising producer of itaconic acid, since
it grows as single cells (yeast-like) in submerged cultivations and it is
extremely robust in high osmotic media and real seawater. Moreover, U. maydis
can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this
fungus combines important advantages of yeasts and filamentous fungi.
Nevertheless, the biomass pretreatment does indeed affect the subsequent
itaconic acid production. Although U. maydis is insusceptible to most possible
impurities from pretreatment, high amounts of salts or residues of organic acids
can slow microbial growth and decrease the production. Consequently, the
pretreatment step needs to fit the prerequisites defined by the actual
microorganisms applied for fermentation. Biotechnologically produced itaconic acid (IA) is a promising organic acid with
a wide range of applications and the potential to open up new application fields
in the area of polymer chemistry, pharmacy, and agriculture. In this study, a
systematic process optimization was performed with an own isolated strain of
Aspergillus terreus and transferred from a 250-mL to a 15-L scale. An IA
concentration of 86.2 g/L was achieved within 7 days with an overall
productivity of 0.51 g/(L h), a maximum productivity of 1.2 g/(L h), and a yield
of 86 mol%. A cultivation of other well-known A. terreus strains with the
developed process showed no significant differences. Based on this, a process is
developed providing a high final IA concentration independent of the used strain
combined with high reproducibility. Cells of Aspergillus terreus, free and immobilized in polyurethane foam, were
employed in itaconic acid fermentation processes on glycerol-based media. The
purpose was to assess their suitability for animal bone char solubilization and
the development of a biotechnological alternative to P fertilizers chemically
produced from rock phosphate. Animal bones constitute a renewable source of P
that can replace the traditionally used finite, nonrenewable rock phosphate as a
P source. Glycerol was an excellent substrate for growth (10.2 g biomass L(-1))
and itaconic acid production (26.9 g L(-1)) by free fungal cells after 120-h
fermentation. Simultaneously, A. terreus solubilized the insoluble phosphate to
a yield of 23 to 50 %, depending on the particle size and concentration.
Polyurethane foam cut into cubes of 0.5-0.6 cm per side, with 0.3 mm pore size
and applied at 2.0 g L(-1) proved to be an excellent cell carrier. In repeated
batch fermentation, the immobilized mycelium showed a high capacity to
solubilize animal bone char, which resulted on average in 168.8 mg L(-1) soluble
phosphate per 48-h cycle and 59.4 % yield (percent of total phosphate)
registered in the fourth batch. Aspergillus niger has an extraordinary potential to produce organic acids as
proven by its application in industrial citric acid production. Previously, it
was shown that expression of the cis-aconitate decarboxylase gene (cadA) from
Aspergillus terreus converted A. niger into an itaconic acid producer (Li et
al., Fungal Genet Bio 48: 602-611, 2011). After some initial steps in production
optimization in the previous research (Li et al., BMC biotechnol 12: 57, 2012),
this research aims at modifying host strains and fermentation conditions to
further improve itaconic acid production. Expression of two previously
identified A. terreus genes encoding putative organic acid transporters (mttA,
mfsA) increased itaconic acid production in an A. niger cis-aconitate
decarboxylase expressing strain. Surprisingly, the production did not increase
further when both transporters were expressed together. Meanwhile, oxalic acid
was accumulated as a by-product in the culture of mfsA transformants. In order
to further increase itaconic acid production and eliminate by-product formation,
the non-acidifying strain D15#26 and the oxaloacetate acetylhydrolase (oahA)
deletion strain AB 1.13 ∆oahA #76 have been analyzed for itaconic acid
production. Whereas cadA expression in AB 1.13 ∆oahA #76 resulted in higher
itaconic acid production than strain CAD 10.1, this was not the case in strain
D15#26. As expected, oxalic acid production was eliminated in both strains. In a
further attempt to increase itaconic acid levels, an improved basal citric
acid-producing strain, N201, was used for cadA expression. A selected
transformant (N201CAD) produced more itaconic acid than strain CAD 10.1, derived
from A. niger strain AB1.13. Subsequently, we have focused on the influence of
dissolved oxygen (D.O.) on itaconic acid production. Interestingly, reduced D.O.
levels (10-25 %) increased itaconic acid production using strain N201 CAD.
Similar results were obtained in strain AB 1.13 CAD + HBD2.5 (HBD 2.5) which
overexpressed a fungal hemoglobin domain. Our results showed that overexpression
of the hemoglobin domain increased itaconic acid production in A. niger at lower
D.O. levels. Evidently, the lower levels of D.O. have a positive influence on
itaconic acid production in A. niger strains. Itaconic acid is an unsaturated dicarbonic acid which has a high potential as a
biochemical building block, because it can be used as a monomer for the
production of a plethora of products including resins, plastics, paints, and
synthetic fibers. Some Aspergillus species, like A. itaconicus and A. terreus,
show the ability to synthesize this organic acid and A. terreus can secrete
significant amounts to the media (>80 g/L). However, compared with the citric
acid production process (titers >200 g/L) the achieved titers are still low and
the overall process is expensive because purified substrates are required for
optimal productivity. Itaconate is formed by the enzymatic activity of a
cis-aconitate decarboxylase (CadA) encoded by the cadA gene in A. terreus.
Cloning of the cadA gene into the citric acid producing fungus A. niger showed
that it is possible to produce itaconic acid also in a different host organism.
This review will describe the current status and recent advances in the
understanding of the molecular processes leading to the biotechnological
production of itaconic acid. |
Which types of cancer can be recognized and treated by the use of immunotherapy? | When normal cells turn into cancer cells, some of the antigens on their surface change. These cells, like many body cells, constantly shed bits of protein from their surface into the circulatory system. Often, tumor antigens are among the shed proteins.
These shed antigens prompt action from immune defenders, including cytotoxic T cells, natural killer cells, and macrophages. According to one theory, patrolling cells of the immune system provide continuous bodywide surveillance, catching and eliminating cells that undergo malignant transformation. Tumors develop when this immune surveillance breaks down or is overwhelmed.
A new approach to cancer therapy uses antibodies that have been specially made to recognize specific cancers such as Melanoma, Leukaemia, Lung Cancer, Colorectal Cancer, Breast Cancer, Head Cancer and Pancreatic Cancer. | Twenty-five patients with metastatic melanoma were treated with a therapeutic
vaccine ("theraccine") consisting of allogeneic melanoma lysates and a novel
adjuvant, DETOX (Ribi ImmunoChem Research, Inc, Hamilton, MT). Each patient
received 200 antigenic units (20 x 10(6) tumor cell equivalents) subcutaneously
on weeks 1, 2, 3, 4, and 6. Clinical responses included one complete remission,
three partial remissions, and a long-term (17-month) stability. Two other
patients had mixed responses, with partial remissions of numerous subcutaneous
nodules. Sites of responsive disease included primarily the skin, but ileal,
breast, and a liver metastasis also responded. Removal of residual lesions in
patients with partial remissions, whose other lesions had disappeared during
treatment, led to long disease-free survivals. The median duration of remission
was 17 months, with four of the five responders alive for at least 24 months
after treatment. An increase in precursors of cytolytic T cells (CTLs)
correlated with clinical outcome, when complete, partial, and mixed responses
and long-term stability were considered. The CTLs recognized melanoma-associated
antigens on many cell lines, but not other types of tumor or normal lymphocytes.
Skin-test reactivity to melanoma antigens and serum antibodies against the
melanoma cells was unrelated to clinical response. Toxicity was minimal,
restricted largely to minor soreness at the site of injection. Only five
patients, four of whom were treated with repeated courses, developed severe
granulomas. These results confirm that active-specific immunization with
allogeneic lysates of melanoma administered with the adjuvant DETOX can induce
immunity to melanoma, and can induce regressions of disease in a proportion of
patients with metastatic disease with little toxicity. The 6th annual Cancer Vaccines and Immunotherapy Colloquium at Walker's Cay was
held under the auspices of the Albert B. Sabin Vaccine Institute on March 10-13,
2004. The Colloquium consisted of a select group of 34 scientists representing
academia, biotechnology and pharmaceutical industry. The main goal of this
gathering was to promote in a peaceful and comfortable environment exchanges
between basic and clinical science. The secondary benefit was to inspire novel
bench to bedside ventures and at the same time provide feed back about promising
and/or disappointing clinical results that could help re-frame some scientific
question or guide the design of future trials. Several topics were covered that
included tumor antigen discovery and validation, platforms for vaccine
development, tolerance, immune suppression and tumor escape mechanisms, adoptive
T cell therapy and dendritic cell-based therapies, clinical trials and
assessment of response. Here we report salient points raised by speakers or by
the audience during animated discussion that followed each individual
presentation. Since the discovery of tumor-associated antigens (TAAs), researchers have tried
to develop immune-based anti-cancer therapies. Thanks to their specificity,
monoclonal antibodies (mAbs) offer the major advantage to induce fewer side
effects than those caused by non-specific conventional treatments (e.g.,
chemotherapy, radiotherapy). Passive immunotherapy by means of mAbs or cytokines
has proved efficacy in oncology and validated the use of immune-based agents as
part of anti-cancer treatment options. The next step was to try to induce an
active immune protection aiming to boost own's host immune defense against TAAs.
Cancer vaccines are thus developed to specifically induce active immune
protection targeting only tumor cells while preserving normal tissues from a
non-specific toxicity. But, as most of TAAs are self antigens, an immune
tolerance against them exists representing a barrier to effective vaccination
against these oncoproteins. One promising approach to break this immune
tolerance consists in the use of anti-idiotypic (anti-Id) mAbs, so called Ab2,
as antigen surrogates. This vaccination strategy allows also immunization
against non-proteic antigens (such as carbohydrates). In some clinical studies,
anti-Id cancer vaccines indeed induced efficient humoral and/or cellular immune
responses associated with clinical benefit. This review article will focus on
recent achievements of anti-Id mAbs use as cancer vaccines in solid tumors. Dendritic cell (DC)-based vaccines with the use of various antigen loading
methods have been developed for cancer immunotherapy. Electroporation (EP) of a
whole tumor cell lysate into DCs was previously found to be more potent for
eliciting antigen-specific CD8 + T-cells compared to co-incubation of tumor cell
lysates with DCs in vitro. In the present report, we studied the feasibility,
safety and antitumor effect in the clinical use of an EP-DC vaccine for the
immunotherapy of various types of human solid tumors. We successfully prepared
an autologous tumor lysate-loaded EP-DC vaccine with high cell viability by the
closed-flow electroporation system. In the phase I clinical trial, mild adverse
events associated with the EP-DC vaccine were found during the treatment of
advanced or recurrent cancer, or during the adjuvant therapy of some types of
cancer; no autoimmune responses were observed after treatment with the
autologous tumor lysate-loaded EP-DC vaccines. For the antitumor effect of the
EP-DC vaccine against the 41 various types of solid tumor, the overall response
rate [complete remission (CR) + partial response (PR)] was 4.9% (2/41) and the
clinical benefit rate [CR+ PR + long stable disease (SD)] was 31.7% (13/41).
Furthermore, the delayed-type hypersensitivity (DTH) reactivity was positive in
most cases of long SD and the positive rate of DTH was 91.7% (11/12) for the
patients with clinical benefit. In conclusion, the safety and feasibility of the
EP-DC vaccine with autologous tumor lysates were confirmed, and it was found
that the antitumor effect might be associated with the immunological response
induced by the EP-DC vaccine for cancer immunotherapy. The ErbB network is dysregulated in many solid tumors. To exploit this, we have
developed a chimeric Ag receptor (CAR) named T1E28z that targets several
pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric
cytokine receptor named 4αβ (combination termed T4), enabling the selective
expansion of engineered T cells using IL-4. Human T4(+) T cells exhibit
antitumor activity against several ErbB(+) cancer types. However, ErbB receptors
are also expressed in several healthy tissues, raising concerns about toxic
potential. In this study, we have evaluated safety of T4 immunotherapy in vivo
using a SCID beige mouse model. We show that the human T1E28z CAR efficiently
recognizes mouse ErbB(+) cells, rendering this species suitable to evaluate
preclinical toxicity. Administration of T4(+) T cells using the i.v. or
intratumoral routes achieves partial tumor regression without clinical or
histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is
accompanied by dose-dependent side effects. Toxicity mediated by T4(+) T cells
results from target recognition in both tumor and healthy tissues, leading to
release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme
cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by
prior macrophage depletion, consistent with clinical data that implicate IL-6 in
this pathogenic event. These data demonstrate that CAR-induced cytokine release
syndrome can be modeled in mice that express target Ag in an appropriate
distribution. Furthermore, our findings argue that ErbB-retargeted T cells can
achieve therapeutic benefit in the absence of unacceptable toxicity, providing
that route of administration and dose are carefully optimized. Dendritic cells (DCs) occupy a privileged position at the interface between
innate and adaptive immunity, orchestrating a large panel of responses to both
physiological and pathological cues. In particular, whereas the presentation of
antigens by immature DCs generally results in the development of immunological
tolerance, mature DCs are capable of priming robust, and hence therapeutically
relevant, adaptive immune responses. In line with this notion, functional
defects in the DC compartment have been shown to etiologically contribute to
pathological conditions including (but perhaps not limited to) infectious
diseases, allergic and autoimmune disorders, graft rejection and cancer. Thus,
the possibility of harnessing the elevated immunological potential of DCs for
anticancer therapy has attracted considerable interest from both researchers and
clinicians over the last decade. Alongside, several methods have been developed
not only to isolate DCs from cancer patients, expand them, load them with
tumor-associated antigens and hence generate highly immunogenic clinical grade
infusion products, but also to directly target DCs in vivo. This intense
experimental effort has culminated in 2010 with the approval by the US FDA of a
DC-based preparation (sipuleucel-T, Provenge®) for the treatment of asymptomatic
or minimally symptomatic metastatic castration-refractory prostate cancer. As an
update to the latest Trial Watch dealing with this exciting field of research
(October 2012), here we summarize recent advances in DC-based anticancer
regimens, covering both high-impact studies that have been published during the
last 13 mo and clinical trials that have been launched in the same period to
assess the antineoplastic potential of this variant of cellular immunotherapy. New York esophageal squamous cell carcinoma-1 (NY-ESO-1), a cancer testis
antigen, is an ideal target for adoptive cell transfer immunotherapy. Evidence
from several clinical trials in melanoma and other maligcies shows the
potential value of targeting the NY-ESO-1 antigen in immune-based therapy of
metastatic tumors. However, the incidence of NY-ESO-1 expression in metastatic
melanoma is unknown, and thus, it is unclear how many patients might benefit
from this therapy. In this study, we analyzed NY-ESO-1 expression in 222
melanoma specimens, including 16 primary and 206 metastatic tumors. Our results
support previous findings showing higher expression of NY-ESO-1 in metastatic
(58/206; 28.2%) versus primary (0/16) tumors. In addition, our results show that
the epithelioid subtype of melanoma has the highest incidence of NY-ESO-1
expression. These findings provide evidence of the value of this specific
adoptive cell transfer therapy for the treatment of metastatic melanoma. High-dose (HD) IL-2 therapy in patients with cancer increases the general
population of Tregs, which are positive for CD4, CD25, and the Treg-specific
marker Foxp3. It is unknown whether specific subsets of Tregs are activated and
expanded during HD IL-2 therapy or whether activation of any particular Treg
subset correlates with clinical outcome. Here, we evaluated Treg population
subsets that were induced in patients with melanoma following HD IL-2 therapy.
We identified a Treg population that was positive for CD4, CD25, Foxp3, and the
inducible T cell costimulator (ICOS). This Treg population increased more than
any other lymphocyte subset during HD IL-2 therapy and had an activated Treg
phenotype, as indicated by high levels of CD39, CD73, and TGF-β. ICOS(+) Tregs
were the most proliferative lymphocyte population in the blood after IL-2
therapy. Patients with melanoma with enhanced expansion of ICOS(+) Tregs in
blood following the first cycle of HD IL-2 therapy had worse clinical outcomes
than patients with fewer ICOS(+) Tregs. However, there was no difference in
total Treg expansion between HD IL-2 responders and nonresponders. These data
suggest that increased expansion of the ICOS(+) Treg population following the
first cycle of HD IL-2 therapy may be predictive of clinical outcome. Cancer testis (CT) antigens are attractive targets for cancer immunotherapy
because their expression is restricted in normal germ line tissues but
frequently detected in variety of tumors. OY-TES-1 is identified as a member of
CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer
(CRC) is solely based on mRNA analysis. None of previous researches has studied
OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was
generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR
and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24
colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC
patients were also tested for OY-TES-1 antibody by ELISA. Our results showed
that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC
(73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and
colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein
expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of
adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was
found in normal colon by either RT-PCR or immunohistochemistry. Furthermore,
OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004)
and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%,
7/73) of CRC patients' sera but not in 76 healthy donors. This finding
demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce
humoral immune response spontaneously in CRC patients, suggesting that it might
be a promising immunotherapy target for CRC. Ganglioside GD2 is highly expressed on neuroectoderm-derived tumors and
sarcomas, including neuroblastoma, retinoblastoma, melanoma, small cell lung
cancer, brain tumors, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma in
children and adolescents, as well as liposarcoma, fibrosarcoma, leiomyosarcoma
and other soft tissue sarcomas in adults. Since GD2 expression in normal tissues
is restricted to the brain, which is inaccessible to circulating antibodies, and
in selected peripheral nerves and melanocytes, it was deemed a suitable target
for systemic tumor immunotherapy. Anti-GD2 antibodies have been actively tested
in clinical trials for neuroblastoma for over the past two decades, with proven
safety and efficacy. The main limitations have been acute pain toxicity
associated with GD2 expression on peripheral nerve fibers and the inability of
antibodies to treat bulky tumor. Several strategies have been developed to
reduce pain toxicity, including bypassing complement activation, using blocking
antibodies, or targeting of O-acetyl-GD2 derivative that is not expressed on
peripheral nerves. To enhance anti-tumor efficacy, anti-GD2 monoclonal
antibodies and fragments have been engineered into immunocytokines,
immunotoxins, antibody drug conjugates, radiolabeled antibodies, targeted
oparticles, T-cell engaging bispecific antibodies, and chimeric antigen
receptors. The challenges of these approaches will be reviewed to build a
perspective for next generation anti-GD2 therapeutics in cancer therapy. The programmed death-1 (PD-1) pathway is important in the maintece of
peripheral tolerance and homeostasis through suppression of T cell receptor
signaling. As such, it is employed by many tumors as a means of immune escape.
We have investigated the role of this pathway in human ovarian cancer (OC) to
assess its potential role as a diagnostic and/or prognostic marker and
therapeutic target, following recent clinical trial success of antibody therapy
directed at this pathway. We show programmed death ligand-1 (PD-L1) expression
on monocytes in the ascites and blood of patients with maligt OC is
strikingly higher than those with benign/borderline disease, with no overlap in
the values between these groups. We characterize the regulation of this molecule
and show a role of IL-10 present in ascitic fluid. Flow cytometric analysis of T
cells present in the ascites and blood showed a correlation of PD-1 expression
with maligt tumors versus benign/borderline, in a similar manner to PD-L1
expression on monocytes. Finally, we demonstrate functional links between PD-L1
expression on monocytes and OC tumor cells with suppression of T cell responses.
Overall, we present data based on samples obtained from women with ovarian
cancer, suggesting the PD-1 pathway may be used as a reliable diagnostic marker
in OC, as well as a viable target for use with PD-1/PD-L1-directed antibody
immunotherapy. Substantial progress has been made in the treatment of precursor B-cell acute
lymphoblastic leukemia (B-ALL), but recurrent disease remains a leading cause of
death in children due to cancer and outcomes for adults with B-ALL remain poor.
Recently, complete clinical responses have been observed in small numbers of
patients with B-ALL treated with adoptive immunotherapy using T cells
genetically engineered to express chimeric antigen receptors (CARs) targeting
CD19, a cell surface molecule present in essentially all cases of B-ALL.
Preclinical data suggest that CARs targeting CD22, another antigen present in
the majority of B-ALL cases, are similarly potent. Several clinical studies
already under way will soon more clearly define the rate of response to this
novel therapy in B-ALL. Further work is needed to identify optimal platforms for
CAR-based adoptive immunotherapy for leukemia, to establish guidelines for
managing toxicity, and to determine whether the remissions induced by this
approach can be rendered durable. BACKGROUND: Post-autologous stem cell transplantation (ASCT) consolidation and
maintece therapies in multiple myeloma (MM) have recently been the central
focus of studies. However, there have been no reports of Japanese patients with
MM treated with post-ASCT consolidation/maintece therapies.
PATIENTS AND METHODS: We retrospectively evaluated eight Japanese patients with
newly-diagnosed symptomatic MM who received ASCT after high-dose melphalan, and
three to four courses of bortezomib-plus-dexamethasone and two courses of
lenalidomide-plus-dexamethasone followed by maintece lenalidomide for 6-24
months.
RESULTS: Four patients achieved complete response (CR) after ASCT, and five
patients (63%) achieved stringent CR after the consolidation and maintece
therapy; two out of these five were in molecular CR. At the median follow-up of
38 months, all patients were alive and only one patient had disease progression
following post-ASCT therapy.
CONCLUSION: Post-ASCT consolidation and maintece therapy using lenalidomide
may be effective in the treatment of Japanese patients with MM. Transgenic rodent models of prostate cancer have served as valuable preclinical
models to evaluate novel treatments and understand maligt disease
progression. In particular, a transgenic rat autochthonous model of prostate
cancer using the SV40 large T antigen expressed under a prostate-specific
probasin promoter was previously developed as a model of androgen-dependent
prostate cancer (TRAP). In the current report, we backcrossed this strain to the
Lewis strain, an inbred rat strain better characterized for immunological
analyses. We demonstrate that Lewis transgenic rats (Lew-TRAP) developed
prostate adenocarcinomas with 100% penetrance by 25 weeks of age. Tumors were
predomitly androgen-dependent, as castration prevented tumor growth in the
majority of animals. Finally, we demonstrate that Lew-TRAP rats could be
immunized with a DNA vaccine encoding a human prostate tumor antigen (prostatic
acid phosphatase) with the development of Lewis strain-specific T-cell
responses. We propose that this Lew-TRAP strain, and prostate tumor cell lines
derived from this strain, can be used as a future prostate cancer immunotherapy
model. The superiority of dendritic cells (DCs) as antigen-presenting cells has been
exploited in numerous clinical trials, where generally monocyte-derived DCs
(Mo-DCs) are injected to induce immunity in patients with cancer or infectious
diseases. Despite promising expansion of antigen-specific T cells, the clinical
responses following vaccination have been limited, indicating that further
improvements of DC vaccine potency are necessary. Pre-clinical studies suggest
that vaccination with combination of primary DC subsets, such as myeloid and
plasmacytoid blood DCs (mDCs and pDCs, respectively), may result in stronger
clinical responses. However, it is a challenge to obtain high enough numbers of
primary DCs for immunotherapy, since their frequency in blood is very low. We
therefore explored the possibility to generate them from hematopoietic
progenitor cells (HPCs). Here, we show that by inhibiting the aryl hydrocarbon
receptor with its antagonist StemRegenin 1 (SR1), clinical-scale numbers of
functional BDCA2(+)BDCA4(+) pDCs, BDCA1(+) mDCs, and BDCA3(+)DNGR1(+) mDCs can
be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were
phenotypically and functionally comparable to peripheral blood DCs. They
secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α,
interleukin (IL)-12, and tumor necrosis factor (TNF)-α and upregulated
co-stimulatory molecules and maturation markers following stimulation with
Toll-like receptor (TLR) ligands. Further, they induced potent allogeneic T-cell
responses and activated antigen-experienced T cells. These findings demonstrate
that SR1 can be exploited to generate high numbers of functional pDCs and mDCs
from CD34(+) HPCs, providing an alternative option to Mo-DCs for immunotherapy
of patients with cancer or infections. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of
maligcy. Via a broad stimulation of the immune system, PDAC activates both
antitumor immune responses and immunosuppressive mechanisms. We propose that new
immunotherapeutic strategies for the management of PDAC should be designed to
specifically neutralize the immunosuppressive tumor microenvironment. CD4+ T cells promote cytotoxic T lymphocyte (CTL)-mediated anticancer immune
responses. We have recently identified ideal tumor-associated antigen
(TAA)-derived long peptides (LPs) that elicit not only TAA-specific TH1
response, but also CTLs, through cross-presentation. The LP-specific TH1 cell
responses were augmented in cancer patients vaccinated with CTL epitopes. Our
findings support the clinical application of LP-based immunotherapy. The tumor microenvironment is a complex assortment of cells that includes a
variety of leukocytes. The overall effect of the microenvironment is to support
the growth of tumors and suppress immune responses. Immunotherapy is a highly
promising form of cancer treatment, but its efficacy can be severely compromised
by an immunosuppressive tumor microenvironment. Chemotherapy and radiation
treatment can mediate tumor reduction through cytotoxic effects, but it is
becoming increasingly clear that these forms of treatment can be used to modify
the tumor microenvironment to liberate tumor antigens and decrease
immunosuppression. Chemotherapy and radiotherapy can be used to modulate the
tumor microenvironment to enhance immunotherapy. |
What is the typical rash associated with gluten ? | Dermatitis herpetiformis is a lifelong, gluten-sensitive, blistering skin disease with pathognomonic immunoglobulin (Ig)A deposits in the papillary dermis. | Twenty-one patients with dermatitis herpetiformis have been on a gluten free
diet regularly followed up for at least one year (mean four years). Eighteen
patients had a 'flat' mucosal appearance (grade III), one patient had moderately
severe mucosal abnormality (grade II), one patient had mild mucosal abnormality
(grade I), and one patient had a normal mucosal appearance (grade O). On the
diet, 10 patients had no skin rash and took no dapsone, seven patients
controlled the skin rash on a lower dose of dapsone, and four noticed no
improvement. There was no correlation between pre-diet jejunal morphology and
response of the skin. A repeat jejunal biopsy, on the gluten free diet, was
possible in 15 patients. While all those with skin improvement showed some
improvement in jejunal morphology, there was no association between the degree
of skin improvement and the degree of recovery of the jejunal mucosa. A study was undertaken to determine whether the skin eruption of linear IgA
disease (LAD) was gluten dependent. Six patients with LAD were treated with a
gluten free diet (GFD) for an average period of 33 months (range 19-48).
Although one patient with LAD had an enteropathy which was clearly gluten
sensitive, there was no convincing evidence that the rash of any of the patients
responded to a GFD. Four of the six patients showed no significant alteration in
their drug requirements. The remaining 2 patients showed a fall in minimum drug
requirement but there was no increase after gluten challenge indicating that
they were entering spontaneous remission. This contrasts to the situation in
dermatitis herpetiformis, where both the rash and the enteropathy are gluten
dependent. These data add further to the evidence that LAD and dermatitis
herpetiformis are separate entities. Twenty one children with dermatitis herpetiformis were studied in an attempt to
evaluate the response in the skin, in jejunal morphology, and in jejunal
immunoglobulin containing cell counts to gluten elimination and subsequent
gluten challenge. In all of the 15 patients whose jejunal biopsy was studied
after the eventual gluten challenge the jejunal lesion had returned in 2.4 to 28
months. The numbers of IgA- and IgM-containing cells were similarly raised in
primary and postchallenge biopsies. In the 13 patients whose skin improved
during a gluten free diet and who were challenged with gluten the rash worsened
and the dapsone/sulphapyridine requirement increased. The jejunal deterioration
was equally marked in the six patients whose gluten challenge was stopped
because of an intractable rash as it was in those who completed the preplanned
challenge. The specimens of the former, however, had significantly more
IgA-containing cells than specimens of the latter. The number of intraepithelial
lymphocytes clearly reflected the degree of intestinal damage. IgA-containing
cells proved to be the most sensitive indicator of an immune reaction taking
place in the gut of these patients. Even in the two children with initially
normal or nearly normal jejunal mucosa, the IgA cell counts in the jejunal
lamina propria were markedly raised. Fifty seven children with dermatitis herpetiformis, 18 from Finland and 39 from
Hungary, were studied. Diagnostic criteria included the finding of granular IgA
deposits in the skin of all patients. The mean age at onset of the rash was 7 X
2 years and favoured sites were the elbows, knees, and buttocks. Symptoms
suggesting small intestinal disease were rare but in 35 (61%) of the children
subtotal villous atrophy and in 16 (28%) partial villous atrophy were found on
jejunal biopsy. Eighteen children underwent a second biopsy after a mean of 21
months on a gluten free diet; villous height was found to be increased and the
intraepithelial lymphocyte count decreased in all these patients. Gluten
challenge caused a reversal in the two children who underwent a third biopsy.
The effect of the gluten free diet on the rash was examined in Finnish children
by observing the daily requirements of dapsone, a drug used to control the rash
at the beginning of the diet. Eight (67%) of the 12 children were able to stop
taking dapsone after a mean of 11 months on the diet and all three patients
treated with diet alone became asymptomatic after three to 6 months on the diet.
These results confirm that most children with dermatitis herpetiformis have
jejunal villous atrophy, though they rarely have gastrointestinal symptoms. The
central role of gluten in childhood dermatitis herpetiformis is evidenced by the
fact that a gluten free diet helps the damaged jejunal mucosa to recover and
controls the rash even in those children who do not have an abnormal jejunal
biopsy. Dermatitis herpetiformis (DH) is a relatively rare skin disorder with an
estimated incidence of 1:10,000 in the UK. It is characterized by urticarial
plaques and blisters on the elbows, buttocks, and knees, although other sites
may also be involved. The eruption tends to be persistent: only 10-15% of
patients have spontaneous remission over a 25-year study period. The disease is
characterized by the presence of IgA deposits in the upper dermis of uninvolved
skin and the diagnosis should not be made in the absence of these deposits.
Two-thirds of patients have a small intestinal enteropathy with villous atrophy
as seen in coeliac disease (CD). However, the remaining third also show evidence
of a gluten sensitivity in the intestine, as judged by increased lymphocytic
infiltration of the epithelium. Villous atrophy also ensues after gluten
challenge in those patients with previous normal villous architecture. The
initial treatment of the rash is with one of the following three drugs, dapsone,
sulphapyridine or sulphamethoxypyridazine. However, the rash also clears with
gluten withdrawal. It must be stressed, however, that the average time to
achieve significant reduction in drug requirements is 6 months and it can be
over 2 years before drugs are no longer required. On re-introduction of gluten
the eruption recurs. Patients with DH have a high incidence of auto-immune
disorders, thyroid disease, pernicious anaemia, and insulin-dependent diabetes,
and should be screened for those diseases on a yearly basis. As with coeliac
disease there is also an increased incidence of lymphoma and a gluten-free diet
appears to protect patients from this complication. The mechanism by which
gluten causes the skin lesions has still to be elucidated, but current
investigations implicate lymphocytes and cytokines in the pathogenesis. The
original hypothesis of an antigen-antibody reaction in the skin with complement
activation causing the skin lesions, may not be correct. Gluten-free diets have been used in the treatment of patients with dermatitis
herpetiformis in our department since 1967. Of the 212 patients with dermatitis
herpetiformis attending between 1967 and 1992, 133 managed to take the diet, and
78 of these achieved complete control of their rash by diet alone. Of the
remaining 55 patients taking a gluten-free diet, all but three were taking
partial diets; over half of these patients managed to substantially reduce the
dose of medication required. Of the 77 patients taking a normal diet, eight
entered spontaneous remission, giving a remission rate of 10%; a further two
patients who had been taking gluten-free diets were found to have remitted when
they resumed normal diets. Loss of IgA from the skin was observed in 10 of 41
(24%) patients taking strict gluten-free diets. These patients had been taking
their diets for an average of 13 years (range 5-24 years), and their rash had
been controlled by diet alone for an average of 10 years (range 3-16 years). The
advantages of a gluten-free diet in the management of patients with dermatitis
herpetiformis are: (i) the need for medication is reduced or abolished; (ii)
there is resolution of the enteropathy, and (iii) patients experience a feeling
of well-being after commencing the diet. Thus, we propose that a gluten-free
diet is the most appropriate treatment for patients with dermatitis
herpetiformis. Serum IgA class antigliadin antibodies (IgA-AGA) are increased in untreated
patients with coeliac disease and dermatitis herpetiformis (DH), and it has been
suggested that salivary IgA-AGA measurements could be used as a non-invasive
screening test for gluten-sensitive enteropathy. In the present study salivary
and serum IgA-AGA were measured by an ELISA test in 10 untreated patients with
DH. The results were compared to IgA-AGA levels in nine patients with DH on a
long-term gluten-free diet (GFD) and in 20 healthy control subjects on an
ordinary diet. The mean serum but not salivary IgA-AGA concentrations were
significantly higher in the untreated than in the patients with DH on a
long-term GFD. When the 10 untreated patients with DH adhered to a GFD for 3
months, the rash disappeared and the mean serum IgA-AGA decreased to normal
levels, but no change was found in the mean salivary IgA-AGA concentration.
These results show that serum but not salivary IgA-AGA measurements are suitable
for monitoring GFD treatment in patients with DH. The discrepancy between the
serum and salivary IgA-AGA concentrations suggests that systemic and salivary
IgA-AGA responses are controlled separately. Dermatitis herpetiformis (DH) is an IgA mediated blistering skin disease
characterized by the presence of granular deposits of IgA in papillary dermis.
The major significant advances in our understanding of DH have been the
demonstration that DH patients also have coeliac diseases (CD) and that the rash
is also gluten dependent. As a result, it is now possible to cure patients by
gluten withdrawal from the diet. The other major significant finding has been
the presence of IgA in the uninvolved, now used as the diagnostic criterion for
the disease. Despite the fact that it has been known for over fifty years that
gluten causes the enteropathy of CD, and for over thirty years the rash of DH,
it is still not known how gluten produces these effects. Future immunological
studies may look at ways of inducing tolerance to gluten peptides once the toxic
ones have been identified. Vaccination against gluten peptides may also be
possible in those affected with gluten sensitive disorders. Gluten sensitive enteropathy has various manifestations, of which the two major
forms are classical coeliac disease (cCD) and dermatitis herpetiformis (DH). In
cCD predomitly the small intestine is affected, whereas in DH also the skin
is affected showing typical rash and IgA deposits. The symptoms in both forms
are dependent on gluten intake. The factors diversifying these two clinical
outcomes are unknown. In the present report we evaluated the role of the major
genetic susceptibility locus, HLA DQ, in 25 families, in which both forms of the
disease, cCD and DH, occurred in siblings. By using the family-based approach it
can be assumed that within each family variation in environmental factors is
substantially lower than in the standard case-control setting, and also the
problems related to population stratification can be avoided. Results from the
Finnish family material with 25 discordant and 85 concordant sib pairs, and from
additional case-control material comprising 71 unrelated Hungarian DH and 68 cCD
patients, together indicated that the HLA DQ locus did not differ between the
two major outcomes of gluten sensitive enteropathy. The non-HLA DR;DQ factors
are critical for the different clinical manifestations of gluten sensitivity. BACKGROUND: A life-long gluten-free diet is the treatment of choice for
dermatitis herpetiformis, which is considered to be coeliac disease of the skin.
OBJECTIVES: To investigate the effects on long-term remission of dermatitis
herpetiformis in patients who underwent a gluten challenge and subsequently
reintroduced dietary gluten.
PATIENTS AND METHODS: We studied 38 patients (14 male and 24 female) with
biopsy-confirmed dermatitis herpetiformis. They had followed a gluten-free diet
for a mean of 8 years, achieving clinical remission and intestinal
normalization. The patients were asked to reintroduce gluten in their diet and
agreed to undergo skin and intestinal biopsies during the follow-up.
RESULTS: Of the 38 patients abandoning a gluten-free diet, 31 reported the onset
of rash within an average of 2 months. Seven subjects (three males, mean age 15
years at challenge) experienced no clinical or histological relapses (median
follow-up 12 years), and lost IgA immunoglobulin from the skin. The two series
of patients differed in terms of age at diagnosis (mean age: 26.6 vs. 6 years),
the use of dapsone (one of 31 vs. four of seven) and adherence to the
gluten-free diet (strict compliance in 26 of 31 vs. none of seven).
CONCLUSIONS: Our data suggest that the ingestion of small doses of gluten in
childhood and/or the use of an anti-inflammatory drug may modify the
immunological response inducing immune tolerance. We report long-term clinical
and histological remissions in seven patients with dermatitis herpetiformis
after the reintroduction of dietary gluten. Dermatitis herpetiformis is characterised by granular IgA precipitates in the
papillary dermis. In contrast to other autoimmune blistering diseases, where
tissue-deposited and circulating autoantibodies recognise the same target within
the skin, in dermatitis herpetiformis a serum IgA reacting with a component of
the healthy papillary dermis has not been detected. Recently, the antigenic
specificity of pathognomic skin-bound IgA has been clarified: the immune
precipitates contain epidermal transglutaminase, an enzyme not previously
detected in the papillary region of normal skin. Furthermore, serum IgA in
dermatitis herpetiformis has been found to bind epidermal transglutaminase.
These findings may relate to the fact, that dermatitis herpetiformis is
associated with gluten sensitive enteropathy, coeliac disease, which is
characterised by IgA type autoantibodies to a closely related enzyme, tissue
transglutaminase. The two transglutaminases are highly homologous, and
therefore, cross reactivity of the two antibodies might explain why patients
with gluten sensitive enteropathy, with or without skin disease, generally have
serum autoantibodies to both enzymes. There is growing evidence that dermatitis
herpetiformis should be considered as the skin manifestation of gluten
sensitivity developing in those patients with mild coeliac disease, who produce
epidermal transglutaminase autoantibodies of high avidity and affinity. Both the
skin and the small bowel diseases are gluten dependent and are strongly
associated with HLA DQ with no genetic differences to explain the two
phenotypes. The question should be asked whether the rash in dermatitis
herpetiformis is a classic autoimmune blistering disease or whether it has an
immune complex basis, which is the most likely alternative. Dermatitis herpetiformis (DH) is an autoimmune blistering skin disorder that is
associated with gluten sensitivity. It presents as a papulovesicular rash and is
often associated with enteropathy. The rash resolves when the patient is placed
on a gluten-free diet and/or dapsone. DH, as well as celiac disease, is tightly
associated with DQ2 and DQ8. A novel mouse model for DH is described that
utilizes the NOD background and the HLA-DQ8 transgene. The addition of DQ8
contributes sensitivity to gliadin, and the addition of the NOD background
contributes to autoimmunity and pathogenesis. Fifteen NOD DQ8+ mice of 90 that
were sensitized to gluten developed blistering pathology similar to that seen in
DH. Neutrophil infiltration of the dermis, deposition of IgA at the
dermal-epidermal junction, and a complete reversal of the blistering phenomenon
with the administration of a gluten-free diet with or without dapsone were
observed. None of the 3 blistering mice examined had small-bowel pathology. This
animal model of DH will be useful to determine the specificity of the IgA
deposits, as well as the pathogenic mechanisms that occur in the skin as a
result of gluten ingestion. Dermatitis herpetiformis and coeliac disease are gluten-sensitive diseases that
share immunopathological mechanisms. Neurological disorders are reported in both
diseases, being more frequent in coeliac disease. Dermatitis herpetiformis is
rare in paediatric populations and only sporadic cases with neurological
dysfunction are reported. Uncertainty exists as to whether early treatment may
stop or reverse neurological symptoms. We describe here the case of a child
presenting with a rash and ataxia, diagnosed with dermatitis herpetiformis, in
whom neurological symptoms and signs regressed after treatment. Dermatitis herpetiformis is an autoimmune blistering disease that appears as a
cutaneous manifestation of gluten intolerance. It is one of a group of disorders
that have gluten sensitivity in common, including celiac disease and gluten
ataxia. Patients with dermatitis herpetiformis present with a pruritic
papulovesicular rash on extensor surfaces and on the buttocks. Immunological
studies demonstrate the presence of specific immunoglobulin (Ig) A
anti-endomysial and anti-transglutaminase antibodies. The finding of granular
deposits of IgA along the dermal-epidermal junction is pathognomonic of
dermatitis herpetiformis. Treatment of dermatitis herpetiformis is based on a
life-long, strict gluten-free diet, which improves all clinical aspects of
gluten sensitivity, and dapsone, a drug that is only effective for the skin
manifestations. Dermatitis herpetiformis (DH) is an autoimmunity-driven inflammatory blistering
dermatosis associated with a gluten-dependent enteropathy. Tissue
transglutaminase (tTG) and nonapeptides of gliadin (npG) are considered in its
pathomechanism/diagnostics. Here, the diagnostic accuracy of anti-tTG/anti-npG
IgA ELISAs in Slavic DH patients with active skin rash was assessed through
creating receiver operating characteristic (ROC) curves, determining cutoff
values, and calculating correlations between levels of anti-tTG/anti-npG IgA in
DH, IgA/neutrophil-mediated non-DH patients and healthy persons. Altogether,
sera from 80 Slavic individuals were examined. There were negligible differences
between cutoff points obtained by the ELISAs manufacturer and those in this
study. There were statistically significant correlations between levels of
anti-tTG/anti-npG IgA in both DH group and the group of IgA/neutrophil-mediated
non-DH dermatoses. There was no such correlation in healthy controls. It seems
that IgA autoantibodies to tTG and npG in the IgA/neutrophil-mediated DH are
produced in the coordinated way implying their causal relationship. |
Which proteins constitute the methyl-directed mismatch repair system (MMR) in bacteria? | The mismatch repair system (MMR) recognizes and corrects mismatched or unpaired bases caused mainly by DNA polymerase, and contributes to the fidelity of DNA replication in living cells. In bacteria, the methyldirected mismatch repair (MMR) is comprised of MutS and MutL proteins, encoded by the mutS/L operon. | Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages
appears to be correlated with their undermethylation during growth in dam+ (GATC
ade-methylase) bacteria. This observation is corroborated by the sequence
analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH
protein of the methyl-directed mismatch repair system recognizes and cleaves the
undermethylated GATC sequences in the course of mismatch repair. To enquire
whether the MutH function of the methyl-directed mismatch repair system
participates in counterselection of GATC sequences in enterobacteriophages, we
have studied the yield of bacteriophage phi X174 containing either 0, 1, or 2
GATC sequences, in wild type, dam, and mut (H, L, S, U) Escherichia coli.
Following transfection with unmethylated DNA containing two GATC sequences, a
net decrease in the yield of infective particles was observed in all bacterial
mutH+ dam- strains, whereas no detectable decrease was observed in bacteria
infected by DNA without GATC sequence. This effect of the MutH function is
maximum in wild type and mutL and mutS bacteria whereas the effect is not
significant in mutU bacteria, suggesting an interaction of the helicase II with
the MutH protein. However, in dam+ bacteria, the presence of GATC sequences
leads to an increased yield of infective particles. The effect of GATC sequence
and its Dam methylation system on phage yield in mutH- bacteria reveals that
methylated GATC sequences are advantageous to the phage.(ABSTRACT TRUNCATED AT
250 WORDS) Some of the molecular aspects of methyl-directed mismatch repair in E. coli have
been characterized. These include: mismatch recognition by mutS protein in which
different mispairs are bound with different affinities; the direct involvement
of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with
strand selection based on methylation of the DNA at those sites. In addition,
communication over a distance between a mismatch and d(GATC) sites has been
implicated. Analysis of mismatch correction in a defined system (Lahue et al.,
unpublished) should provide a direct means to further molecular aspects of this
process. Mismatch repair genes are involved in increasing the fidelity of replication by
specific repair of DNA polymerase incorporation errors. In Escherichia coli, the
best studied mismatch repair (MMR) pathway is the methyl-directed long patch
repair system which is mediated by three gene products; MutS, MutL and MutH.
These are conserved in higher eukaryotes. Mutations in human homologues of these
proteins have been shown to be implicated in hereditary non-polyposis colorectal
cancer (HNPCC). Alterations in the coding regions of MMR genes result in a
mutator phenotype with marked instability of microsatellite sequences,
indicative of a deficiency in DNA repair. The expansion of normally polymorphic CTG microsatellites in certain human genes
has been identified as the causative mutation of a number of hereditary
neurological disorders, including Huntington's disease and myotonic dystrophy.
Here, we have investigated the effect of methyl-directed mismatch repair (MMR)
on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations
and find two opposing effects. In contrast to orientation-dependent repeat
instability in wild-type E. coli and yeast, we observed no orientation
dependence in MMR- E. coli cells and suggest that, for the repeat that we have
studied, orientation dependence in wild-type cells is mainly caused by
functional mismatch repair genes. Our results imply that slipped structures are
generated during replication, causing single triplet expansions and contractions
in MMR- cells, because they are left unrepaired. On the other hand, we find that
the repair of such slipped structures by the MMR system can go awry, resulting
in large contractions. We show that these mutS-dependent contractions arise
preferentially when the CTG sequence is encoded by the lagging strand. The
nature of this orientation dependence argues that the small slipped structures
that are recognized by the MMR system are formed primarily on the lagging strand
of the replication fork. It also suggests that, in the presence of functional
MMR, removal of 3 bp slipped structures causes the formation of larger
contractions that are probably the result of secondary structure formation by
the CTG sequence. We rationalize the opposing effects of MMR on repeat tract
stability with a model that accounts for CTG repeat instability and loss of
orientation dependence in MMR- cells. Our work resolves a contradiction between
opposing claims in the literature of both stabilizing and destabilizing effects
of MMR on CTG repeat instability in E. coli. In Escherichia coli and related bacteria, the very-short-patch (VSP) repair
pathway uses an endonuclease, Vsr, to correct T-G mismatches that result from
the deamination of 5-methylcytosines in DNA to C-G. The products of mutS and
mutL, which are required for adenine methylation-directed mismatch repair (MMR),
enhance VSP repair. Multicopy plasmids carrying mutS alleles that are domit
negative for MMR were tested for their effects on VSP repair. Some mutS
mutations (class I) did not lower VSP repair in a mutS(+) background, and most
class I mutations increased VSP repair in mutS cells more than plasmids
containing mutS(+). Other plasmid-borne mutS mutations (class II) and mutS(+)
decreased VSP repair in the mutS(+) background. Thus, MutS protein lacking
functions required for MMR can still participate in VSP repair, and our results
are consistent with a model in which MutS binds transiently to the mispair and
then translocates away from the mispair to create a specialized structure that
enhances the binding of Vsr. We have recently described the presence of a high proportion of Pseudomonas
aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the
lungs of cystic fibrosis (CF) patients. In four out of 11 independent P.
aeruginosa strains, the high mutation frequency was found to be complemented
with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the
cloning and sequencing of two additional P. aeruginosa mismatch repair genes and
the characterization, by complementation of deficient strains, of these two
putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe
the alterations in the mutS, mutL and uvrD genes responsible for the mutator
phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven
out of the 11 mutator strains were found to be defective in the MMR system (four
mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes
contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion
after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between
two eight nucleotide direct repeats. This deletion, involving domain II of MutS,
was found to be the main one responsible for mutS inactivation. The second mutL
strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL,
a residue known to be essential for its ATPase activity. Finally, the uvrD
strain had three amino acid substitutions within the conserved ATP binding site
of the deduced UvrD polypeptide, showing defective mismatch repair activity.
Interestingly, cells carrying this mutant allele exhibited a fully active
UvrABC-mediated excision repair. The results shown here indicate that the
putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that
their alteration results in a mutator phenotype. In Escherichia coli and related enteric bacteria, repair of base-base mismatches
is performed by two overlapping biochemical processes, methyl-directed mismatch
repair (MMR) and very short-patch (VSP) repair. While MMR repairs replication
errors, VSP repair corrects to C*G mispairs created by 5-methylcytosine
deamination to T. The efficiency of the two pathways changes during the
bacterial life cycle; MMR is more efficient during exponential growth and VSP
repair is more efficient during the stationary phase. VSP repair and MMR share
two proteins, MutS and MutL, and although the two repair pathways are not
equally dependent on these proteins, their dual use creates a competition within
the cells between the repair processes. The structural and biochemical data on
the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays
a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic
studies of the two repair pathways have helped eliminate certain models for MMR
and put restrictions on models that can be developed regarding either repair
process. We review here recent information about the biochemistry of both repair
processes and describe the balancing act performed by cells to optimize the
competing processes during different phases of the bacterial life cycle. A simple genetic system has been developed to test the effect of over-expression
of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on
methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on
detection of Lac(+) revertants using MMR-proficient or MMR-deficient E. coli
strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We
report that expression of wild-type hMLH1 protein causes an approx. 19-fold
increase in mutation rates. The mutator phenotype was due to the ability of
hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby
interfering with the formation of complexes between MMR proteins and mismatched
DNA. Conversely, expression of proteins encoded by alleles deriving from
hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates,
depending on the specific amino acid substitutions. These effects parallel the
MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated
proteins. Bacteria contain a number of error prevention and error correction systems that
maintain genome stability. However, strains exhibiting elevated mutation
frequencies have recently been reported amongst natural populations of
pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa,
Neisseria meningitidis, Helicobacter pylori and Streptococcus pneumoniae. The
majority of naturally occurring, strong mutators contain defects in the
methyl-directed mismatch repair (MMR) system, with mutations in mutS
predominating. MMR-deficient strains possess superior genetic backgrounds for
the selection of some antibiotic-resistance mutations since mutation frequencies
up to 1000-fold higher than normal strains have been reported, and resistance
levels achieved in mutators can be greater than those arising in non-mutator
hosts. MMR is a major constraint to interspecies recombination events. Removal
of this barrier, as in the case of MMR defective mutators, also enhances the
frequency of horizontal gene transfer, which is an important mechanism of
acquired drug resistance in bacteria. Permanent global mutator status is
associated with loss of fitness as mutators accumulate deleterious mutations
more frequently than non-mutators. Fitness limitations of mutators may be
overcome simply by the high bacterial cell densities that can be achieved during
acute infection or by the adoption of transient mutator status. Mutators are a
risk factor during the treatment of bacterial infections as they appear to
enhance the selection of mutants expressing high- and low-level antibiotic
resistance and have the capacity to refine existing plasmid-located resistance
determits. Recombinogenic engineering methodology, also known as recombineering, utilizes
homologous recombination to create targeted changes in cellular DNA with great
specificity and flexibility. In Escherichia coli, the Red recombination system
from bacteriophage lambda has been used successfully to modify both plasmid and
chromosomal DNA in a highly efficient manner, using either a linear
double-stranded DNA fragment or a synthetic single-stranded oligonucleotide
(SSO). The current model for Red/SSO-mediated recombination involves the SSO
first annealing to a transient, single-stranded region of DNA before being
incorporated into the chromosome or plasmid target. It has been observed
previously, in both eukaryotes and prokaryotes, that mutations in the two
strands of the DNA double helix are 'corrected' by complementary SSOs with
differing efficiencies. Here we investigate further the factors that influence
the strand bias as well as the overall efficiency of Red/SSO-mediated
recombination in E.coli. We show that the direction of DNA replication and the
nature of the SSO-encoded mismatch are the main factors dictating the
recombinational strand bias. However, the influence that the SSO-encoded
mismatch exerts upon the recombinational strand bias is abolished in E.coli
strains that are defective in mismatch repair (MMR). This reflects the fact that
different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway
with differing efficiencies. Furthermore, our data indicate that transcription
has negligible influence on the strand bias. These results demonstrate for the
first time that the interplay between DNA replication and MMR has a major effect
on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli. The mismatch repair system (MMR) recognizes and corrects mismatched or unpaired
bases caused mainly by DNA polymerase, and contributes to the fidelity of DNA
replication in living cells. In Escherichia coli, the MutHLS system is known to
function in MMR, and homologues of MutS and MutL are widely conserved in almost
all organisms. However, the MutH endonuclease has not been found in the majority
of organisms. Such organisms, including Thermus thermophilus HB8, often possess
the so-called MutS2 protein, which is highly homologous to MutS but contains an
extra C-terminal stretch. To elucidate the function of MutS2, we overexpressed
and purified T. thermophilus MutS2 (ttMutS2). ttMutS2 demonstrated the ability
to bind double-stranded (ds) DNA, but, unlike ttMutS, ttMutS2 showed no
specificity for mismatched duplexes. ttMutS2 ATPase activity was also detected
and was stimulated by dsDNA. Our results also showed that ttMutS2 incises dsDNA.
ttMutS2 incises not only oligo dsDNA but also plasmid DNA, suggesting that
ttMutS2 possesses an endonuclease activity. At low concentrations, the incision
activity was not retained, but was promoted by T. thermophilus MutL. We have characterized the mismatch repair system (MMR) of the highly
radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show
that the MMR system is functional in this organism, where it participates in
ensuring the fidelity of DNA replication and recombination. The system relies on
the activity of two key proteins, MutS1 and MutL, which constitute a conserved
core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in
a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a
ten-fold increase in the efficiency of integration of a donor point-mutation
marker during bacterial transformation. Inactivation of the mismatch
repair-associated UvrD helicase increased the level of spontaneous mutagenesis,
but had no effect on marker integration--suggesting that binding of MutS1 and
MutL proteins to a mismatched heteroduplex suffices to inhibit recombination
between non identical (homeologous) DNAs. In contrast, inactivation of MutS2,
encoded by the second mutS -related gene present in D. radiodurans, had no
effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins
were as resistant to gamma-rays, mitomycin C and UV-irradiation as wild-type
bacteria, suggesting that the mismatch repair system is not essential for the
reconstitution of a functional genome after DNA damage. One of the popular ideas is that decline in methyl-directed mismatch repair
(MMR) in carbon-starved bacteria might facilitate occurrence of stationary-phase
mutations. We compared the frequency of accumulation of stationary-phase
mutations in carbon-starved Pseudomonas putida wild-type and MMR-defective
strains and found that knockout of MMR system increased significantly emergence
of base substitutions in starving P. putida. At the same time, the appearance of
1-bp deletion mutations was less affected by MMR in this bacterium. The spectrum
of base substitution mutations which occurred in starving populations of P.
putida wild-type strain was distinct from mutation spectrum identified in
MMR-defective strains. The spectrum of base substitutions differed also in this
case when mutants emerged in starved populations of MutS or MutL-defective
strains were comparatively analyzed. Based on our results we suppose that other
mechanisms than malfunctioning of MMR system in resting cells might be
considered to explain the accumulation of stationary-phase mutations in P.
putida. To further characterize populations of P. putida starved on selective
plates, we stained bacteria with LIVE/DEAD kit in situ on agar plates. We found
that although the overall number of colony forming units (CFU) did not decline
in long-term-starved populations, these populations were very heterogeneous on
the plates and contained many dead cells. Our results imply that slow growth of
subpopulation of cells at the expenses of dead cells on selective plates might
be important for the generation of stationary-phase mutations in P. putida.
Additionally, the different survival patterns of P. putida on the same selective
plates hint that competitive interactions taking place under conditions of
prolonged starvation of microbial populations on semi-solid surfaces might be
more complicated than previously assumed. The Escherichia coli DNA Mismatch Repair (MMR) protein MutS exist as dimers and
tetramers in solution, and the identification of its functional oligomeric state
has been matter of extensive study. In the present work, we have analyzed the
oligomerization state of MutS from Pseudomonas aeruginosa a bacterial species
devoid of Dam methylation and MutH homologue. By analyzing native MutS and
different mutated versions of the protein, we determined that P. aeruginosa MutS
is mainly tetrameric in solution and that its oligomerization capacity is
conducted as in E. coli, by the C-terminal region of the protein. The analysis
of mismatch oligonucleotide binding activity showed that wild-type MutS binds to
DNA as tetramer. The DNA binding activity decreased when the C-terminal region
was deleted (MutSDelta798) or when a full-length MutS with tetramerization
defects (MutSR842E) was tested. The ATPase activity of MutSDelta798 was similar
to MutSR842E and diminished respect to the wild-type protein. Experiments
carried out on a P. aeruginosa mutS strain to test the proficiency of different
oligomeric versions of MutS to function in vivo showed that MutSDelta798 is not
functional and that full-length dimeric version MutSR842E, is not capable of
completely restoring the MMR activity of the mutant strain. Additional
experiments carried out in conditions of high mutation rate induced by the base
analogue 2-AP confirm that the dimeric version of MutS is not as efficient as
the tetrameric wild-type protein to prevent mutations. Therefore, it is
concluded that although dimeric MutS is sufficient for MMR activity, optimal
activity is obtained with the tetrameric version of the protein and therefore it
should be considered as the active form of MutS in P. aeruginosa. MutL is required to assist the mismatch repair protein MutS during initiation of
the methyl-directed mismatch repair (MMR) response in various organisms ranging
from prokaryotes to eukaryotes. Despite this necessity, the inherent propensity
of MutL to aggregate has led to significant difficulties in determining its
biological relationship with other MMR-related proteins. Here, we perform
analysis on the thermostable MutL protein found in Thermotoga maritima MSB8
(TmL). Size exclusion chromatographic analysis indicates the lack of aggregated
forms with the exception of a dimeric TmL. Small-angle X-ray scattering (SAXS)
analysis reveals that the solution structures of the full-length TmL and its
corresponding complexes with nucleotides and ssDNA undergo conformational
changes. The elucidated TmL SAXS model is superimposed to the crystal structure
of the C-terminal domain of Escherichia coli MutL. In addition, the N-terminal
SAXS model of TmL exists as monomeric form, indicating that TmL has a
structurally flexible N-terminal domain. TmL SAXS analysis can suggest a
considerable possibility on a new 3D view of the previously unresolved
full-length MutL molecule. The methyl-directed mismatch repair (MMR) mechanism has been extensively studied
in vitro and in vivo, but one of the difficulties in determining the biological
relationships between the MMR-related proteins is the tendency of MutL to
self-aggregate. The properties of a stable MutL homologue were investigated
using a thermostable MutL (TmL) from Thermotoga maritima MSB8 and whose size
exclusion chromatographic and crosslinking analyses were compatible with a
dimeric form of TmL. TmL underwent conformational changes in the presence of
nucleotides and single-stranded DNA (ssDNA) with ATP binding not requiring ssDNA
binding activity of TmL, while ADPnP-stimulated TmL showed a high ssDNA binding
affinity. Finally, TmL interacted with the T. maritima MutS (TmS), increasing
the affinity of TmS to mismatched DNA base pairs and suggesting that the role of
TmL in the formation of a mismatched DNA-TmS complex may be a pivotal
observation for the study of the initial MMR system. [BMB reports 2009; 42(1):
53-58]. DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways
involved in the repair of T:G mismatches. To learn about competition and
cooperation between these two repair pathways, we analyzed the physical and
functional interaction between MutL and Vsr using biophysical and biochemical
methods. Analytical ultracentrifugation reveals a nucleotide-dependent
interaction between Vsr and the N-terminal domain of MutL. Using chemical
crosslinking, we mapped the interaction site of MutL for Vsr to a region between
the N-terminal domains similar to that described before for the interaction
between MutL and the strand discrimination endonuclease MutH of the MMR system.
Competition between MutH and Vsr for binding to MutL resulted in inhibition of
the mismatch-provoked MutS- and MutL-dependent activation of MutH, which
explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and
VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a
MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the
enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile
MutS-MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or
at the time of, activation of downstream effector molecules such as Vsr or MutH. O(6)-alkylG adducts are highly mutagenic due to their capacity to efficiently
form O(6)-alkylG:T mispairs during replication, thus triggering G→A transitions.
Mutagenesis is largely prevented by repair strategies such as reversal by
alkyltransferases or excision by nucleotide excision repair (NER). Moreover,
methyl-directed mismatch repair (MMR) is known to trigger sensitivity to
methylating agents via a mechanism that involves recognition by MutS of the
O(6)-mG:T replication intermediates. We wanted to investigate the mechanism by
which MMR controls the genotoxicity of environmentally relevant O(6)-alkylG
adducts formed by ethylene oxide and propylene oxide. Recently, the
alkyltransferase-like gene ybaZ (eATL) was shown to enhance repair of these
slightly larger O(6)-alkylG adducts by NER. We analyzed the toxicity and
mutagenesis induced by these O(6)-alkylG adducts using single-adducted plasmid
probes. We show that the eATL gene product prevents MMR-mediated attack of the
O(6)-alkylG:T replication intermediate for the larger alkyl groups but not for
methyl. In vivo data are compatible with the occurrence of repeated cycles of
MMR attack of the O(6)-alkylG:T intermediate. In addition, in vitro, the eATL
protein efficiently prevents binding of MutS to the O(6)-alkylG:T mispairs
formed by the larger alkyl groups but not by methyl. In conclusion, eATL not
only enhances the efficiency of repair of these larger adducts by NER, it also
shields these adducts from MMR-mediated toxicity. Stress-promoted mutations that occur in nondividing cells (adaptive mutations)
have been implicated strongly in causing genetic variability as well as in
species survival and evolutionary processes. Oxidative stress-induced DNA damage
has been associated with generation of adaptive His(+) and Met(+) but not Leu(+)
revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we
report that an interplay between MutY and MutSL (mismatch repair system [MMR])
plays a pivotal role in the production of adaptive Leu(+) revertants.
Essentially, the genetic disruption of MutY dramatically reduced the reversion
frequency to the leu allele in this model system. Moreover, the increased rate
of adaptive Leu(+) revertants produced by a MutSL knockout strain was
significantly diminished following mutY disruption. Interestingly, although the
expression of mutY took place during growth and stationary phase and was not
under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon
increased the expression of mutY several times. Thus, in starved cells,
saturation of the MMR system may induce the expression of mutY, disturbing the
balance between MutY and MMR proteins and aiding in the production of types of
mutations detected by reversion to leucine prototrophy. In conclusion, our
results support the idea that MMR regulation of the mutagenic/antimutagenic
properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells. DNA mismatch repair (MMR) systems can be classified as either MutH-dependent or
MutH-independent. In bacteria, extensive studies have been conducted with the
MutH-dependent MMR in Escherichia coli and its close relatives. The picture of
MutH-independent MMR in other bacteria is less clear, as MMR components other
than MutS and MutL have not been identified in the majority of bacteria.
Bacillus anthracis is one of the MutH-less Gram(+) bacteria in the phylum of
Firmicutes. We used papillation as a tool to search for B. anthracis new mutator
strains and identified a spontaneous mutator that carries a minitransposon
insertion in the BAS4289 locus. The mutational frequency and specificity
exhibited in this mutant were comparable to that of MMR-deficient strains with
knockouts of mutL or mutS. It retained a similar UV sensitivity profile as that
of the wild type. BAS4289 encodes a putative DNA helicase RecD2 that shares 30%
sequence identity with Deinococcus radiodurans RecD2, a well characterized
superfamily 1B helicase whose homologs are widely present in Firmicutes complete
genomes. We demonstrated that the N-terminal region of RecD2, a unique sequence
extension used to distinguish RecD2 from RecD1, was important for B. anthracis
RecD2, as mutations in the N-terminal conserved motifs affected its DNA repair
function. This is the first report of a RecD2 helicase being associated with
MMR. RecD2 and our recently described YycJ protein are likely to be two
additional components in the B. anthracis MutH-independent MMR system. Type 1 fimbriae and flagella, two surface organelles critical for colonization
of the urinary tract by uropathogenic Escherichia coli (UPEC), mediate opposing
virulence objectives. Type 1 fimbriae facilitate adhesion to mucosal cells and
promote bacterial persistence in the urinary tract, while flagella propel
bacteria through urine and along mucous layers during ascension to the upper
urinary tract. Using a transposon screen of the E. coli CFT073 fim locked-ON
(L-ON) mutant, a construct that constitutively expresses type 1 fimbriae and
represses motility, we identified six mutants that exhibited a partial
restoration of motility. Among these six mutated genes was mutS, which encodes a
component of the methyl-directed mismatch repair (MMR) system. When complemented
with mutS in trans, motility was again repressed. To determine whether the MMR
system, in general, is involved in this reciprocal control, we characterized the
effects of gene deletions of other MMR components on UPEC motility. Isogenic
deletions of mutS, mutH, and mutL were constructed in both wild-type CFT073 and
fim L-ON backgrounds. All MMR mutants showed an increase in motility in the
wild-type background, and ΔmutH and ΔmutS mutations increased motility in the
fim L-ON background. Cochallenge of the wild-type strain with an MMR-defective
strain showed a subtle but significant competitive advantage in the bladder and
spleen for the MMR mutant using the murine model of ascending urinary tract
infection after 48 h. Our findings demonstrate that the MMR system generally
affects the reciprocal regulation of motility and adherence and thus could
contribute to UPEC pathogenesis during urinary tract infections. Mismatch repair (MMR) increases the fidelity of DNA replication by identifying
and correcting replication errors. Processivity clamps are vital components of
DNA replication and MMR, yet the mechanism and extent to which they participate
in MMR remains unclear. We investigated the role of the Bacillus subtilis
processivity clamp DnaN, and found that it serves as a platform for mismatch
detection and coupling of repair to DNA replication. By visualizing functional
MutS fluorescent fusions in vivo, we find that MutS forms foci independent of
mismatch detection at sites of replication (i.e. the replisome). These MutS foci
are directed to the replisome by DnaN clamp zones that aid mismatch detection by
targeting the search to nascent DNA. Following mismatch detection, MutS
disengages from the replisome, facilitating repair. We tested the functional
importance of DnaN-mediated mismatch detection for MMR, and found that it
accounts for 90% of repair. This high dependence on DnaN can be bypassed by
increasing MutS concentration within the cell, indicating a secondary mode of
detection in vivo whereby MutS directly finds mismatches without associating
with the replisome. Overall, our results provide new insight into the mechanism
by which DnaN couples mismatch recognition to DNA replication in living cells. The mechanism of DNA replication is one of the driving forces of genome
evolution. Bacterial DNA polymerase III, the primary complex of DNA replication,
consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria,
especially in the Firmicutes with low GC content, whereas DnaE is widely
conserved in most Gram-negative and Gram-positive bacteria. PolC contains two
domains, the 3'-5'exonuclease domain and the polymerase domain, while DnaE only
possesses the polymerase domain. Accordingly, DnaE does not have the
proofreading function; in Escherichia coli, another enzyme DnaQ performs this
function. In most bacteria, the fidelity of DNA replication is maintained by
3'-5' exonuclease and a mismatch repair (MMR) system. However, we found that
most Actinobacteria (a group of Gram-positive bacteria with high GC content)
appear to have lost the MMR system and chromosomes may be replicated by
DnaE-type DNA polymerase III with DnaQ-like 3'-5' exonuclease. We tested the
mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found
that the wild type strain is AT-biased while the mutS-deletant strain is
remarkably GC-biased. If we presume that DnaE tends to make mistakes that
increase GC content, these results can be explained by the mutS deletion (i.e.,
deletion of the MMR system). Thus, we propose that GC content is regulated by
DNA polymerase and MMR system, and the absence of polC genes, which participate
in the MMR system, may be the reason for the increase of GC content in
Gram-positive bacteria such as Actinobacteria. |
Is aganglionic megacolon a feature of Down syndrome? | Down syndrome (DS) is recognized by characteristic facial features, intellectual disability, and an increased risk for cardiac malformations and duodenal atresia. Recently, Hirschsprung disease (HSCR), or congenital aganglionic megacolon, has been seen more often among patients with DS. | Hirschsprung disease, or congenital aganglionic megacolon, is commonly assumed
to be a sex-modified multifactorial trait. To test this hypothesis, complex
segregation analysis was performed on data on 487 probands and their families.
Demographic information on probands and the recurrence risk to relatives of
probands are presented. An increased sex ratio (3.9 male:female) and an elevated
risk to sibs (4%), as compared with the population incidence (0.02%), are
observed, with the sex ratio decreasing and the recurrence risk to sibs
increasing as the aganglionosis becomes more extensive. Down syndrome was found
at an increased frequency among affected individuals but not among their
unaffected sibs, and the increase was not associated with maternal age. Complex
segregation analysis was performed on these family data. The families were
classified into separate categories by extent of aganglionosis. For cases with
aganglionosis beyond the sigmoid colon, the mode of inheritance is compatible
with a domit gene with incomplete penetrance, while for cases with
aganglionosis extending no farther than the sigmoid colon, the inheritance
pattern is equally likely to be either multifactorial or due to a recessive gene
with very low penetrance. A model of gene action with random effects during
morphogenesis is compatible with our observations. |
Which is the vector of Louping ill virus? | Louping ill virus (LIV) belongs to the mammalian tick-borne virus group of the genus Flavivirus which cause central nervous system disease. LIV infects the red grouse Lagopus lagopus scoticus, causing high mortality. LIV is transmitted by the tick Ixodes ricinus. | We have constructed recombit baculoviruses and vaccinia viruses containing
cloned DNA, encoding either the envelope protein alone or all of the structural
proteins (core, membrane and envelope) of louping ill virus. Glycosylated viral
envelope protein, presented both inside and on the surface of insect and
mammalian cells, was expressed by all four recombit viruses. Differences in
antigenic presentation of the envelope protein were observed between the
envelope protein and structural protein constructs as well as between the insect
and mammalian cell expression systems. Despite the expression of epitopes known
to elicit neutralizing and protective antibodies when present in authentic
antigen, the recombit envelope protein expressed by either vector failed to
induce, in mice or rabbits, either neutralizing or protective antibodies against
louping ill virus. A single-chain antibody fragment that identifies a neutralizing epitope on the
envelope protein of louping ill and some other tick-borne flaviviruses was
previously expressed in soluble form from bacteria and shown to be functionally
active in vitro. To see whether or not the single-chain antibody could bind and
inactivate infectious virus in vivo, we have used recombit Sindbis virus as a
delivery vehicle for intracellular expression of the antibody fragment. The
variable genes and interchain linker encoding the single-chain antibody were
cloned into a double subgenomic Sindbis virus expression vector to generate
recombit Sindbis virus. Infection with this recombit Sindbis virus
provided high-level cytoplasmic expression of the antibody fragment in mammalian
cells. We demonstrate (i) that the antibody fragment was antigen binding and
(ii) that louping ill virus infectivity was significantly reduced in the
presence of intracellular antibody expressed by the superinfecting recombit
Sindbis virus. This study has examined the efficacy following intramuscular administration of a
recombit Semliki Forest virus (rSFV) vaccine, encoding the prME and NS1
proteins of louping ill virus (LIV), in sheep. Administration of rSFV-LIV
vaccine resulted in transient detection at the injection site and draining lymph
node only and no dissemination to distal sites. In addition, the recombit
vaccine offered complete protection against subcutaneous challenge with LIV, and
partial protection following intranasal administration of LIV. Protected animals
had no pathological changes normally associated with LIV infection, and had
developed high antibody titres. In contrast, the two animals not protected
exhibited classical clinical signs and neuropathological lesions of LIV
infection. These findings indicate that rSFV-based vaccines have the potential
to be developed as effective prototype vaccines for LIV. Repeated predictions that vector-borne disease prevalence will increase with
global warming are usually based on univariate models. To accommodate the full
range of constraints, the present-day distribution of tick-borne encephalitis
virus (TBEv) was matched statistically to current climatic variables, to provide
a multivariate description of present-day areas of disease risk. This was then
applied to outputs of a general circulation model that predicts how climatic
variables may change in the future, and future distributions of TBEv were
predicted for them. The expected summer rise in temperature and decrease in
moisture appears to drive the distribution of TBEv into higher-latitude and
higher-altitude regions progressively through the 2020s, 2050s and 2080s. The
final toe-hold in the 2080s may be confined to a small part of Scandinavia,
including new foci in southern Finland. The reason for this apparent contraction
of the range of TBEv is that its transmission cycles depend on a particular
pattern of tick seasonal dynamics, which may be disrupted by climate change. The
observed marked increase in incidence of tick-borne encephalitis in most parts
of Europe since 1993 may be due to non-biological causes, such as political and
sociological changes. Tick-Borne Encephalitis virus (TBEV) causes dangerous central nervous system
diseases in humans. General infection leads to the development of meningitis or
encephalitis, which is characterized by swelling of the brain due to
inflammation. Tetracyclines may act locally to moderate inflammation in the CNS.
In this study, we investigated the potential clinical benefits of administering
tetracycline hydrochloride to patients hospitalized due to suspected TBEV
infection presenting with fever and evidence of a recent tick bite. We also
characterized an acute immune response to TBEV by profiling certain cytokines
and soluble receptors in Tetracycline-treated and untreated patients. Increased
serum levels of TNF-alpha, IL-1 alpha and IL-6 were found in all patients at
admission. Soluble receptors presented in the serum of patients in a magnitude
higher levels than the corresponding cytokines and were increasing during first
weak of hospitalization. Levels of IL-10 were also rising during that period. In
our study tetracycline hydrochloride acted as an immunomodulator, which was able
to reduce manifestations of inflammation response during TBE course; this action
led to quicker improvement of symptoms and, consequently, to a faster clinical
recovery. The positive result of tetracycline hydrochloride treatment was
accompanied by certain particularities in the dynamics of studied cytokines and
receptors: the concentrations of IL-6, IL-1 beta, TNF-alpha dropped quicker and
reached lower levels, and the concentrations of sIL-6R, IL-1RA, sTNFR1 increased
faster and reached higher maximum levels in the tetracycline-treated groups.
Children had the highest levels of IL-6, which were not neurotoxic. This article describes the steps taken to verify that the tick Ixodes ricinus is
the main vector of Slovenian meningo-encephalitis.In 1954, several unsuccessful
attempts were made to isolate the virus from unengorged ticks collected at
random, but in the following year this system was abandoned, and instead the
search for specimens was carried out on the basis of evidence supplied by
meningo-encephalitis patients with a tick-bite case history.In June-the peak
endemic period-140 unengorged ticks were collected from a part of a forest in
central Slovenia where one of these patients had been working. The ticks were
washed in alcohol, finely ground, and centrifuged, and the supernatant was
inoculated intracerebrally into suckling mice, and intraperitoneally into adult
mice and guinea-pigs. The infected suckling mice died between the fourteenth and
eighteenth day after inoculation; a suspension of their brains was inoculated
intracerebrally into a further batch of suckling and adult mice, which
subsequently fell ill, showing signs in the central nervous system.The virus
strain obtained was subjected to neutralization and complement-fixation tests,
in order to identify the infective agent. Since the recognition that louping-ill, known for well over 100 years as an
epizootic disease of sheep in Scotland, was caused by a virus transmitted by
arthropods, many other arthropod-borne viruses capable of causing encephalitis
in domestic animals or man have been discovered. The author reviews here the
knowledge at present available on these viruses, originally termed
"arthropod-borne encephalitides viruses" but now often referred to as "arbor
viruses".In this discussion of the host and vector relationships of the two
broad groups of arbor viruses - the mosquito-borne and the tick-borne-and of the
distribution, epidemiology and control of the various diseases they cause, the
author includes an outline of the types of investigation likely to provide the
most useful information, stressing in this connexion the value of ecological
surveys. For pathogens transmitted by biting vectors, one of the fundamental assumptions
is often that vector bites are the sole or main route of host infection. Here,
we demonstrate experimentally a transmission route whereby hosts (red grouse,
Lagopus lagopus scoticus) became infected with a member of the tick-borne
encephalitis virus complex, louping ill virus, after eating the infected tick
vector. Furthermore, we estimated from field observations that this mode of
infection could account for 73-98% of all virus infections in wild red grouse in
their first season. This has potential implications for the understanding of
other biting vector-borne pathogens where hosts may ingest vectors through
foraging or grooming. The complex pathogen-host-vector system of the tick-borne louping-ill virus
causes economic losses to sheep and red grouse in upland United Kingdom. This
paper examines the spatial distribution, incidence and effect of control
measures on louping-ill virus in the Bowland Fells of Lancashire. Seroprevalence
in sheep at the beginning of the study varied within the area and was affected
significantly by the frequency of acaricide treatment. There was a clear
decrease over 5 years in the effective force of infection on farms implementing
a vaccination programme, irrespective of acaricide treatment regime, however,
only one third of farms apparently eliminated infection. On farms where
vaccination did not occur or where vaccination was carried out intermittently,
the estimated force of infection was variable or possibly increased. Thus, as
befits a complex host-pathogen system, reductions in prevalence were not as
dramatic as predicted; we discuss the potential explanations for these
observations. The infectivity of flavivirus particles depends on a maturation process that is
triggered by the proteolytic cleavage of the precursor of the M protein (prM).
This activation cleavage is naturally performed by ubiquitous cellular proteases
of the furin family, which typically recognize the multibasic sequence motif
R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus
(TBEV) mutant with an altered cleavage motif, R-X-R, produced immature,
noninfectious particles that could be activated by exogenous trypsin, which
cleaves after single basic residues. Here, we report the adaptation of this
mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid
residues. Using selection pressure in cell culture, two different mutations
conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one
of these mutations (Ser85Phe) occurred three positions upstream of the natural
cleavage site. The other mutation (Arg89His) arose at the natural cleavage
position but involved a His residue, which is not a typical chymotrypsin
cleavage site. Efficient cleavage of protein prM and activation by the
heterologous protease were confirmed using various recombit TBEV mutants.
Mutants with only the originally selected mutations exhibited unimpaired export
kinetics and were genotypically stable during at least six cell culture
passages. However, in contrast to the wild-type virus or trypsin-dependent
mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice.
Our results demonstrate that flaviviruses with altered protease specificities
can be generated and suggest that this approach can be used for the construction
of viral mutants or vectors that can be activated on demand and have restricted
tissue tropism and virulence. Tick-borne flaviviruses are maintained in nature in an enzootic cycle involving
a tick vector and a vertebrate host. Thus, the virus replicates in two disparate
hosts, each providing selective pressures that can influence virus replication
and pathogenicity. To identify viral determits associated with replication in
the individual hosts, plaque purified Langat virus (TP21pp) was adapted to
growth in mouse or tick cell lines to generate two virus variants, MNBp20 and
ISEp20, respectively. Virus adaptation to mouse cells resulted in four amino
acid changes in MNBp20 relative to TP21pp, occurring in E, NS4A and NS4B. A
comparison between TP21pp and ISEp20 revealed three amino acid modifications in
M, NS3 and NS4A of ISEp20. ISEp20, but not MNBp20, was attenuated following
intraperitoneal inoculation of mice. Following isolation from mice brains,
additional mutations reproducibly emerged in E and NS3 of ISEp20 that were
possibly compensatory for the initial adaptation to tick cells. Thus, our data
implicate a role for E, M, NS3, NS4A and NS4B in host adaptation and
pathogenicity of tick-borne flaviviruses. The impact of climate change on vector-borne infectious diseases is currently
controversial. In Europe the primary arthropod vectors of zoonotic diseases are
ticks, which transmit Borrelia burgdorferi sensu lato (the agent of Lyme
disease), tick-borne encephalitis virus and louping ill virus between humans,
livestock and wildlife. Ixodes ricinus ticks and reported tick-borne disease
cases are currently increasing in the UK. Theories for this include climate
change and increasing host abundance. This study aimed to test how I. ricinus
tick abundance might be influenced by climate change in Scotland by using
altitudinal gradients as a proxy, while also taking into account the effects of
hosts, vegetation and weather effects. It was predicted that tick abundance
would be higher at lower altitudes (i.e. warmer climates) and increase with host
abundance. Surveys were conducted on nine hills in Scotland, all of open
moorland habitat. Tick abundance was positively associated with deer abundance,
but even after taking this into account, there was a strong negative association
of ticks with altitude. This was probably a real climate effect, with
temperature (and humidity, i.e. saturation deficit) most likely playing an
important role. It could be inferred that ticks may become more abundant at
higher altitudes in response to climate warming. This has potential implications
for pathogen prevalence such as louping ill virus if tick numbers increase at
elevations where competent transmission hosts (red grouse Lagopus lagopus
scoticus and mountain hares Lepus timidus) occur in higher numbers. In July 2008, owners of seasonal camps in Vermont and Maine were exposed to
large numbers of questing ticks after opening their camps for the season.
Examination of collected specimens revealed that the camp in Vermont was
infested with Ixodes cookei Packard, and the camp in Maine was infested with
Ixodes marxi Banks. In both instances, numerous tick bites were reported by
residents. Both camps were also occupied by wildlife during the off-season,
primarily squirrels (Maine) and skunks (Vermont). Subsequent samples from the
Vermont site were tested for the presence of Powassan encephalitis virus, though
no viral activity was detected. Tick-borne encephalitis virus (TBEV) is a flavivirus with major impact on global
health. The geographical TBEV distribution is expanding, thus making it pivotal
to further characterize the natural virus populations. In this study, we
completed the earlier partial sequencing of a TBEV pulled out of a pool of RNA
extracted from 115 ticks collected on Torö in the Stockholm archipelago. The
total RNA was sufficient for all sequencing of a TBEV genome (Torö-2003),
without conventional enrichment procedures such as cell culturing or suckling
mice amplification. To our knowledge, this is the first time that the genome of
TBEV has been sequenced directly from an arthropod reservoir. The Torö-2003
sequence has been characterized and compared with other TBE viruses. In silico
analyses of secondary RNA structures formed by the two untranslated regions
revealed a temperature-sensitive structural shift between a closed replicative
form and an open AUG accessible form, analogous to a recently described
bacterial thermoswitch. Additionally, novel phylogenetic conserved structures
were identified in the variable part of the 3'-untranslated region, and their
sequence and structure similarity when compared with earlier identified
structures suggests an enhancing function on virus replication and translation.
We propose that the thermo-switch mechanism may explain the low TBEV prevalence
often observed in environmentally sampled ticks. Finally, we were able to detect
variations that help in the understanding of virus adaptations to varied
environmental temperatures and mammalian hosts through a comparative approach
that compares RNA folding dynamics between strains with different mammalian cell
passage histories. A total of 875 nymphal and adult Ixodes ricinus ticks and 148 adult Dermacentor
reticulatus ticks were collected by flagging lower vegetation in the Lublin
region (eastern Poland) and examined for the presence of RNA of tick-borne
encephalitis virus (TBEV) by nested RT-PCR. The minimum infection rate of I.
ricinus ticks with TBEV amounted to 1.6% while the infection rate of D.
reticulatus ticks was 10.8%. The results suggest that D. reticulatus may be a
potential vector of TBEV in Central Europe. BACKGROUND: Ixodes ricinus, a competent vector of several pathogens, is the tick
species most frequently reported to bite humans in Europe. The majority of human
cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the
north-eastern region of Italy. The aims of this study were to detect the
occurrence of endemic and emergent pathogens in north-eastern Italy using adult
tick screening, and to identify areas at risk of pathogen transmission. Based on
our results, different strategies for tick collection and pathogen screening and
their relative costs were evaluated and discussed.
METHODS: From 2006 to 2008 adult ticks were collected in 31 sites and
molecularly screened for the detection of pathogens previously reported in the
same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia
spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results
of this survey, three sampling strategies were evaluated a-posteriori, and the
impact of each strategy on the final results and the overall cost reductions
were analyzed. The strategies were as follows: tick collection throughout the
year and testing of female ticks only (strategy A); collection from April to
June and testing of all adult ticks (strategy B); collection from April to June
and testing of female ticks only (strategy C).
RESULTS: Eleven pathogens were detected in 77 out of 193 ticks collected in 14
sites. The most common microorganisms detected were Borrelia burgdorferi sensu
lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%).
Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B.
garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less
prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A.
phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%).
Co-infections by more than one pathogen were diagnosed in 22% of infected ticks.
The prevalences of infection assessed using the three alternative strategies
were in accordance with the initial results, with 13, 11, and 10 out of 14 sites
showing occurrence of at least one pathogen, respectively. The strategies A, B,
and C proposed herein would allow to reduce the original costs of sampling and
laboratory analyses by one third, half, and two thirds, respectively. Strategy B
was demonstrated to represent the most cost-effective choice, offering a
substantial reduction of costs, as well as reliable results.
CONCLUSIONS: Monitoring of tick-borne diseases is expensive, particularly in
areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can
support the choice of the best monitoring strategy, which should take into
account the ecology of the area under investigation, as well as the available
budget. Parasite-mediated apparent competition occurs when one species affects another
through the action of a shared parasite. One way of controlling the parasite in
the more susceptible host is to manage the reservoir host. Culling can cause
issues in terms of ethics and biodiversity impacts, therefore we ask: can
treating, as compared to culling, a wildlife host protect a target species from
the shared parasite? We used Susceptible Infected Recovered (SIR) models
parameterized for the tick-borne louping ill virus (LIV) system. Deer are the
key hosts of the vector (Ixodes ricinus) that transmits LIV to red grouse
Lagopus lagopus scoticus, causing high mortality. The model was run under
scenarios of varying acaricide efficacy and deer densities. The model predicted
that treating deer can increase grouse density through controlling ticks and
LIV, if acaricide efficacies are high and deer densities low. Comparing deer
treated with 70% acaricide efficacy with a 70% cull rate suggested that
treatment may be more effective than culling if initial deer densities are high.
Our results will help inform tick control policies, optimize the targeting of
control methods and identify conditions where host management is most likely to
succeed. Our approach is applicable to other host-vector-pathogen systems. |
Does HuR protein regulate the splicing process? | HuR and TIA1/TIAL1 are involved in regulation of alternative splicing of SIRT1 pre-mRNA | Regulated gene expression at the post-transcriptional level in higher eukaryotes
is based on a network of interactions among RNA-binding proteins (RBPs)
operating within multifactorial ribonucleoprotein (RNP) complexes, notably
heterogeneous nuclear ribonucleoprotein (hnRNP) and mRNP complexes. We are
interested in interactions involving hnRNP proteins participating in several
steps of mRNA processing (mainly pre-mRNA splicing) and HuR with an established
role in stability/translation of associated mRNAs. hnRNP and HuR proteins have a
major nucleoplasmic localization and ability to shuttle between nucleus and
cytoplasm. We report here on interactions between hnRNP and HuR proteins that
were identified in the context of isolated hnRNP and mRNP complexes. This was
done by the application of immunoprecipitation and pull-down assays on different
sub-cellular fractions prepared from cells of human and mouse origin, as well as
in vivo localization studies. A range of specific associations of HuR with the
shuttling hnRNP A1 and A3 and the non-shuttling hnRNP C1/C2 was identified and
ascribed discrete properties with respect to stability to RNase A and increasing
salt, as well as to cellular distribution. The likelihood of a biological
relevance of these associations was tested under heat shock conditions in
growing cells, which appeared to affect both the sub-nuclear distribution and
interaction of HuR with hnRNPs. The establishment of an extensive association of
HuR with hnRNP components of nuclear hnRNP/mRNP and cytoplasmic mRNP complexes
supports its broader participation in mRNA processing events than initially
anticipated. The ubiquitously expressed RNA-binding protein Hu antigen R (HuR) or ELAVL1 is
implicated in a variety of biological processes as well as being linked with a
number of diseases, including cancer. Despite a great deal of prior
investigation into HuR, there is still much to learn about its function. We take
an important step in this direction by conducting cross-linking and
immunoprecipitation and RNA sequencing experiments followed by an extensive
computational analysis to determine the characteristics of the HuR binding site
and impact on the transcriptome. We reveal that HuR targets predomitly
uracil-rich single-stranded stretches of varying size, with a strong
conservation of structure and sequence composition. Despite the fact that HuR
sites are observed in intronic regions, our data do not support a role for HuR
in regulating splicing. HuR sites in 3'-UTRs overlap extensively with predicted
microRNA target sites, suggesting interplay between the functions of HuR and
microRNAs. Network analysis showed that identified targets containing HuR
binding sites in the 3' UTR are highly interconnected. Poliovirus protease 2A (2A(pro)) obstructs host gene expression by reprogramming
transcriptional and post-transcriptional regulatory events during infection.
Here we demonstrate that expression of 2A(pro) induces a selective
nucleo-cytoplasm translocation of several important RNA binding proteins and
splicing factors. Subcellular fractionation studies, together with
immunofluorescence microscopy revealed an asymmetric distribution of HuR and
TIA1/TIAR in 2A(pro) expressing cells, which modulates splicing of the human Fas
exon 6. Consistent with this result, knockdown of HuR or overexpression of
TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro)-expressing cells. Therefore,
poliovirus 2A(pro) can target alternative pre-mRNA splicing by regulating
protein shuttling between the nucleus and the cytoplasm. SIRT1 is a pleiotropic protein that plays critical and multifunctional roles in
metabolism, senescence, longevity, stress-responses, and cancer, and has become
an important therapeutic target across a range of diseases. Recent research
demonstrated that SIRT1 pre-mRNA undergoes alternative splicing to produce
different isoforms, such as SIRT1 full-length and SIRT1-∆Exon8 variants.
Previous studies revealed these SIRT1 mRNA splice variants convey different
characteristics and functions to the protein, which may in turn explain the
multifunctional roles of SIRT1. However, the mechanisms underlying the
regulation of SIRT1 alternative splicing remain to be elucidated. Our objective
is to search for new pathways that regulate of SIRT1 alternative splicing. Here
we describe experiments showing that HuR and TIA1/TIAL1, two kinds of
RNA-binding proteins, were involved in the regulation of alternative splicing of
SIRT1 pre-mRNA under normal and stress circumstances: HuR increased SIRT1-∆Exon8
by promoting SIRT1 exon 8 exclusion, whereas TIA1/TIAL1 inhibition of the exon 8
exclusion led to a decrease in SIRT1-∆Exon8 mRNA levels. This study provides
novel insight into how the alternative splicing of SIRT1 pre-mRNA is regulated,
which has fundamental implications for understanding the critical and
multifunctional roles of SIRT1. |
Is Titin the largest single protein molecule found in Nature? | Titin, is definitely the largest protein in the body, with a molecular weight of 3 million Dalton and composed of 27,000 amino acids. Titin is the largest protein known to date and acts as a mechanosensor that regulates muscle protein expression in a sarcomere strain-dependent fashion. | Titin is at present the largest known protein (M(r) 3000 kDa) and its expression
is restricted to vertebrate striated muscle. Single molecules span from M- to
Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five
distant titin A-band epitopes, extended their sequences and determined 30 kb
(1000 kDa) of the primary structure of titin. Sequences near the M-line encode a
kinase domain and are closely related to the C-terminus of twitchin from
Caenorhabditis elegans. This suggests that the function of this region in the
titin/twitchin family is conserved throughout the animal kingdom. All other
A-band sequences consist of 100 amino acid (aa) repeats predicting
immunoglobulin-C2 and fibronectin type III globular domains. These domains are
arranged into highly ordered 11 domain super-repeat patterns likely to match the
myosin helix repeat in the thick filament. Expressed titin fragments bind to the
LMM part of myosin and C-protein. Binding strength increases with the number of
domains involved, indicating a cumulative effect of multiple binding sites for
myosin along the titin molecule. We conclude that A-band titin is likely to be
involved in the ordered assembly of the vertebrate thick filament. Titin, the largest protein identified to date (over 1 micron long, almost 3
million daltons in mass) is the third most abundant component of the sarcomere.
In the mature myofibril, titin molecules span from M line to Z line, forming a
third filament system which provides sarcomeric alignment and elastic recoil. In
the developing sarcomere, accumulating evidence from studies both in vivo and in
vitro implicates titin as part of a morphogenetic scaffolding, upon which
critical events in myofibrillogenesis are coordinated in a time- and
space-dependent manner. Titin is the largest polypeptide yet described (relative molecular mass
approximately 3 x 10(6); refs 1, 2) and an abundant protein of striated muscle.
Its molecules are string-like and in vivo span from the M to Z-lines. I-band
regions of titin are thought to make elastic connections between the thick
filament and the Z-line, thereby forming a third type of sarcomere filament.
These would centre the A-band in the sarcomere and provide structural continuity
in relaxed myofibrils. The A-band region of titin seems to be bound to the thick
filament, where it has been proposed to act as a 'molecular ruler' regulating
filament length and assembly. Here, we show that partial titin complementary
DNAs encode a regular pattern of two types of 100-residue motif, each of which
probably folds into a separate domain type. Such motifs are present in several
evolutionarily divergent muscle proteins, all of which are likely to interact
with myosin. One or both of the domain types is therefore likely to bind to
myosin. Titin, the biggest single (poly) peptide found in humans, and throughout nature
so far, was long considered as a good candidate for inherited muscle diseases.
However, disease-causing defects were not known until recently, when this
central sarcomeric protein was associated with human skeletal tibial muscular
dystrophy (TMD/LGMD2J), dilated cardiomyopathy (DCM) and hypertrophic
cardiomyopathy (HCM). Several mutations in different parts of titin have now
been identified and more are expected. Spontaneous mouse and zebrafish mutants
have also been reported. Experimental knock-outs are not viable, even in cases
where just a c-terminal part of the gene was silenced, telling something of the
basic importance of titin for life. In this article we review the current known
structure and functions of this elementary molecule with some emphasis on the
only defects so far known to cause human skeletal muscle disease, mutations in
the c-terminal M-line part of titin. Titin is the largest protein known, and is essential for organising muscle
sarcomeres. It has many domains with a variety of functions, and stretches from
the Z-line to the M-line in the muscle sarcomere. Close to the M-line, titin
contains a kinase domain, which is known to phosphorylate the Z-line protein
telethonin in developing muscle (Mayans, O., van der Ven, P. F., Wilm, M., Mues,
A., Young, P., Furst, D. O., Wilmanns, M. and Gautel, M. (1998) Nature 395,
863-869). This phosphorylation is thought to be important for initiating or
regulating myofibrillogenesis. We used a gene-targeting approach in cultured
myoblasts to truncate the titin gene so that the kinase domain and other domains
downstream of the kinase were not expressed. We recovered cells in which one
allele was targeted. We found that these cells expressed both the full-length
and a truncated titin that was approximately 0.2 MDa smaller than the
corresponding band from wild-type cells. Myofibrillogenesis in these cells was
impaired, in that the myotubes were shorter, and the organisation of the muscle
sarcomeres, M- and Z-lines was poorer than in wild-type cells. There was also an
overall reduction in levels of titin and skeletal myosin expression. These
results suggest that the activity of the titin kinase domain and downstream
sequence are important in organising myofibrils both at the M- and the Z-line
early in myofibrillogenesis. The basis of the Frank-Starling mechanism of the heart is the increase in active
force when muscle is stretched. Various findings have shown that muscle length,
i.e., sarcomere length (SL), modulates activation of cardiac myofilaments at a
given concentration of Ca2+ ([Ca2+]). This augmented Ca2+ activation with SL,
commonly known as "length-dependent activation", is manifested as the leftward
shift of the force-pCa (= -log [Ca2+]) relation as well as by the increase in
maximal Ca2+ -activated force. Despite the numerous studies that have been
undertaken, the molecular mechanism(s) of length-dependent activation is (are)
still not fully understood. The giant sarcomere protein titin/connectin is the
largest protein known to date. Titin/connectin is responsible for most passive
force in vertebrate striated muscle and also functions as a molecular scaffold
during myofibrillogenesis. Recent studies suggest that titin/connectin plays an
important role in length-dependent activation by sensing stretch and promoting
actomyosin interaction. Here we review and extend this previous work and focus
on the mechanism by which titin/connectin might modulate actomyosin interaction. Titin, the largest protein known to date, has been linked to sarcomere assembly
and function through its elastic adaptor and signaling domains. Titin's M-line
region contains a unique kinase domain that has been proposed to regulate
sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a
titin M line-deficient mouse to show that the initial assembly of the sarcomere
does not depend on titin's M-line region or the phosphorylation of T-cap by the
titin kinase. Rather, titin's M-line region is required to form a continuous
titin filament and to provide mechanical stability of the embryonic sarcomere.
Even without titin integrating into the M band, sarcomeres show proper spacing
and alignment of Z discs and M bands but fail to grow laterally and ultimately
disassemble. The comparison of disassembly in the developing and mature knockout
sarcomere suggests diverse functions for titin's M line in embryonic development
and the adult heart that not only involve the differential expression of titin
isoforms but also of titin-binding proteins. Titin is the largest protein in mammals; it forms an elastic filament along the
myofibril of cardiac and skeletal muscles. Novel studies employing the recently
available varied technologies have revealed the molecular mechanisms by which
titin generates passive force in the sarcomere in response to external stretch.
Changes in titin stiffness occur during heart disease via a shift in the
expression ratio of the two main titin isoforms, called N2B (stiff type) and
N2BA (compliant type) titins. Protein kinase (PK)A, PKG and PKC phosphorylate
the cardiac specific I-band titin segment, resulting in an acute decrease (by
PKA and PKG) or increase (by PKC) in passive force. It has also been discovered
that titin performs roles that go beyond passive force generation, by enhancing
or terminating active force production, thereby adjusting the Frank-Starling
mechanism of the heart. Therefore, titin is a self-adjustable and
multi-functional spring that is indispensable for proper heart functions. Here,
we discuss how titin regulates the passive and active properties of cardiac
muscle in normal physiological conditions as well as in chronic heart disease. Titin, the largest protein in the human body, is well known as a molecular
spring in muscle cells and scaffold protein aiding myofibrillar assembly.
However, recent evidence has established another important role for titin: that
of a regulatory node integrating, and perhaps coordinating, diverse signaling
pathways, particularly in cardiomyocytes. We review key findings within this
emerging field, including those related to phosphorylation of the titin springs,
and also discuss how titin participates in hypertrophic gene regulation and
protein quality control. |
what is the role of MEF-2 in cardiomyocyte differentiation? | The myocyte enhancer factor-2 (MEF2) proteins are MADS-box transcription factors that are essential for differentiation of all muscle lineages but their mechanisms of action remain largely undefined. MEF2C expression initiates cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2-5, GATA-4, cardiac alpha-actin, and myosin heavy chain expression. Inactivation of the MEF2C gene causes cardiac developmental arrest and severe downregulation of a number of cardiac markers including atrial natriuretic factor (ANF). BMP-2, a regulator of cardiac development during embryogenesis, was shown to increase PI 3-kinase activity in cardiac precursor cells, resulting in increased expression of sarcomeric myosin heavy chain (MHC) and MEF-2A. Furthermore, expression of MEF-2A increased MHC expression in a PI 3-kinase-dependent manner. Other studies showed that Gli2 and MEF2C proteins form a complex, capable of synergizing on cardiomyogenesis-related promoters. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. These findings show that MEF-2 can direct early stages of cell differentiation into a cardiomyogenic pathway. | The Nkx2-5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic
expression in frog and zebrafish embryos results in an enlarged myocardium;
however, expression of Nkx2-5 in fibroblasts was not able to trigger the
development of beating cardiac muscle. In order to examine the ability of Nkx2-5
to modulate endogenous cardiac specific gene expression in cells undergoing
early stages of differentiation, P19 cell lines overexpressing Nkx2-5 were
differentiated in the absence of Me2SO. Nkx2-5 expression induced
cardiomyogenesis in these cultures aggregated without Me2SO. During
differentiation into cardiac muscle, Nkx2-5 expression resulted in the
activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In
order to compare the abilities of Nkx2-5 and MEF2C to induce cellular
differentiation, P19 cells overexpressing MEF2C were aggregated in the absence
of Me2SO. Similar to Nkx2-5, MEF2C expression initiated cardiomyogenesis,
resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4,
Nkx2-5, GATA-4, cardiac alpha-actin, and myosin heavy chain expression. These
findings indicate the presence of a positive regulatory network between Nkx2-5
and MEF2C and show that both factors can direct early stages of cell
differentiation into a cardiomyogenic pathway. The myocyte enhancer factor-2 (MEF2) proteins are MADS-box transcription factors
that are essential for differentiation of all muscle lineages but their
mechanisms of action remain largely undefined. In mammals, the earliest site of
MEF2 expression is the heart where the MEF2C isoform is detectable as early as
embryonic day 7.5. Inactivation of the MEF2C gene causes cardiac developmental
arrest and severe downregulation of a number of cardiac markers including atrial
natriuretic factor (ANF). However, most of these promoters contain no or low
affinity MEF2 binding sites and they are not significantly activated by any MEF2
proteins in heterologous cells suggesting a dependence on a cardiac-enriched
cofactor for MEF2 action. We provide evidence that MEF2 proteins are recruited
to target promoters by the cell-specific GATA transcription factors, and that
MEF2 potentiates the transcriptional activity of this family of
tissue-restricted zinc finger proteins. Functional MEF2/GATA-4 synergy involves
physical interaction between the MEF2 DNA-binding domain and the carboxy zinc
finger of GATA-4 and requires the activation domains of both proteins. However,
neither MEF2 binding sites nor MEF2 DNA binding capacity are required for
transcriptional synergy. The results unravel a novel pathway for transcriptional
regulation by MEF2 and provide a molecular paradigm for elucidating the
mechanisms of action of MEF2 in muscle and non-muscle cells. The growth and differentiation factor bone morphogenetic protein-2 (BMP-2)
regulates cardiac development during vertebrate embryogenesis. In cardiac
precursor cells, BMP-2 has recently been shown to induce expression of cardiac
transcription factors, including myocyte enhancer factor 2A (MEF-2A). The
specific signal transduction mechanism by which BMP-2 regulates these actions is
not known. We investigated the role of phosphatidylinositol (PI) 3-kinase in
regulating these processes in cardiomyocyte precursor CL6 cells. BMP-2 increased
PI 3-kinase activity in these cells in a time-dependent manner, resulting in
increased expression of sarcomeric myosin heavy chain (MHC) and MEF-2A.
Inhibition of PI 3-kinase abolished these actions of BMP-2, indicating the
involvement of PI 3-kinase in these processes. Furthermore, BMP-2 stimulated
specific protein.DNA complex formation when an MEF-2 DNA recognition element was
used as probe. Antibody supershift assay confirmed the presence of MEF-2A in
this protein.DNA complex. Inhibition of PI 3-kinase activity completely
prevented the MEF-2A.DNA complex formation. BMP-2 also increased transcription
of a reporter gene driven by an MEF-2-specific DNA element in a PI
3-kinase-dependent manner. Ectopic expression of MEF-2A increased BMP-2
transcription to the same extent induced by BMP-2, indicating that MEF-2A may
participate in BMP-2 autoregulation in CL6 cells. Expression of domit
negative PI 3-kinase completely abolished BMP-2-induced as well as
MEF-2A-mediated BMP-2 transcription. Furthermore expression of MEF-2A increased
MHC expression in a PI 3-kinase-dependent manner. Together these data provide
the first evidence that BMP-2-induced PI 3-kinase signaling regulates MEF-2A
expression and define a mechanism of MEF-2A-dependent BMP-2 transcription. The transcription factors Gli2 (glioma-associated factor 2), which is a
transactivator of Sonic Hedgehog (Shh) signalling, and myocyte enhancer factor
2C (MEF2C) play important roles in the development of embryonic heart muscle and
enhance cardiomyogenesis in stem cells. Although the physiological importance of
Shh signalling and MEF2 factors in heart development is well known, the
mechanistic understanding of their roles is unclear. Here, we demonstrate that
Gli2 and MEF2C activated each other's expression while enhancing
cardiomyogenesis in differentiating P19 EC cells. Furthermore, domit-negative
mutant proteins of either Gli2 or MEF2C repressed each other's expression, while
impairing cardiomyogenesis in P19 EC cells. In addition, chromatin
immunoprecipitation (ChIP) revealed association of Gli2 to the Mef2c gene, and
of MEF2C to the Gli2 gene in differentiating P19 cells. Finally,
co-immunoprecipitation studies showed that Gli2 and MEF2C proteins formed a
complex, capable of synergizing on cardiomyogenesis-related promoters containing
both Gli- and MEF2-binding elements. We propose a model whereby Gli2 and MEF2C
bind each other's regulatory elements, activate each other's expression and form
a protein complex that synergistically activates transcription, enhancing
cardiac muscle development. This model links Shh signalling to MEF2C function
during cardiomyogenesis and offers mechanistic insight into their in vivo
functions. The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates
gene expression during cardiomyocyte differentiation and adaptation of the
myocardium to stress. MEF2 activity is enhanced by increasing its transcription
and by MAPK-dependent phosphorylation, and is reduced by binding to class-II
Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here
we show that MEF2 protein abundance is regulated at the translational level,
determining myocyte size, during hypertrophy. In order to reduce MEF2 protein
expression, its silencing through RNA interference required serum deprivation
and, even in this condition, MEF2 protein abundance recovered to basal levels in
presence of phenylephrine. Hypertrophic agonist stimulation of neonatal
ventricular cardiomyocytes increased Mef2 expression by enhancing its
translation, without changing its transcription or blocking degradation of the
protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and
was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA
levels. Calcineurin activity influenced expression of Polypyrimidine Tract
Protein (PTB), contributing to MEF2 translation. Thus, our results show a
previously unrecognized but relevant level of MEF2 activity regulation through
the control of its translation that involves Calcineurin and PTB. The calcium/calmodulin-dependent protein phosphatase calcineurin is required for
the induction of transcriptional events that initiate and promote myogenic
differentiation. An important effector for calcineurin in striated muscle is the
transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the
enzyme and substrate to specific intracellular compartments by scaffold proteins
often confers specificity in phosphatase activity. We now show that the
scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in
myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be
isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2
association is dependent on mAKAP expression. We have identified a peptide
comprising the calcineurin binding domain in mAKAP that can disrupt the binding
of the phosphatase to the scaffold in vivo. Domit interference of
calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity
seen during myoblast differentiation, as well as the expression of endogenous
MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in
cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together
these data illustrate the importance of calcineurin anchoring by the mAKAP
scaffold for MEF2 regulation. |
Which are the main components of mTORC1? | The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), and mLST8. | The mTOR (mammalian target of rapamycin) protein kinase is an important
regulator of cell growth. Two complexes of mTOR have been identified: complex 1,
consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed
mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion
of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K
(S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB
(protein kinase B) at its hydrophobic motif (Ser473). In the present study, we
report that widely expressed isoforms of unstudied proteins termed Protor-1
(protein observed with Rictor-1) and Protor-2 interact with Rictor and are
components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or
Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not
Raptor, a specific component of mTORC1. We show that detergents such as Triton
X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1,
Sin1 and Rictor. We also provide evidence that Rictor regulates the expression
of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2
subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2,
but further work is required to establish its role. Insulin and amino acids act independently to stimulate protein synthesis in
skeletal muscle of neonatal pigs, and the responses decrease with development.
The purpose of this study was to compare the separate effects of fed levels of
INS and AA on the activation of signaling components leading to translation
initiation and how these responses change with development. Overnight-fasted 6-
(n = 4/group) and 26-day-old (n = 6/ group) pigs were studied during 1)
euinsulinemic-euglycemiceuaminoacidemic conditions (controls), 2)
euinsulinemic-euglycemichyperaminoacidemic clamps (AA), and 3)
hyperinsulinemic-euglycemic-euaminoacidemic clamps (INS). INS, but not AA,
increased the phosphorylation of protein kinase B (PKB) and tuberous sclerosis 2
(TSC2). Both INS and AA increased protein synthesis and the phosphorylation of
mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase-1, and
eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), and these
responses were higher in 6-day-old compared with 26-day-old pigs. Both INS and
AA decreased the binding of 4E-BP1 to eIF4E and increased eIF4E binding to
eIF4G; these effects were greater in 6-day-old than in 26-day-old pigs. Neither
INS nor AA altered the composition of mTORC1 (raptor, mTOR, and GbetaL) or
mTORC2 (rictor, mTOR, and GbetaL) complexes. Furthermore, neither INS, AA, nor
age had any effect on the abundance of Rheb and the phosphorylation of
AMP-activated protein kinase and eukaryotic elongation factor 2. Our results
suggest that the activation by insulin and amino acids of signaling components
leading to translation initiation is developmentally regulated and parallels the
developmental decline in protein synthesis in skeletal muscle of neonatal pigs. The mammalian target of rapamycin (mTOR) is part of two distinct complexes,
mTORC1, containing raptor and mLST8, and mTORC2, containing rictor, mLST8 and
sin1. Although great endeavors have already been made to elucidate the function
and regulation of mTOR, the cytoplasmic nuclear distribution of the mTOR
complexes is unknown. Upon establishment of the proper experimental conditions,
we found mTOR, mLST8, rictor and sin1 to be less abundant in the nucleus than in
the cytoplasm of non-transformed, non-immortalized, diploid human primary
fibroblasts. Although raptor is also high abundant in the nucleus, the
mTOR/raptor complex is predomitly cytoplasmic, whereas the mTOR/rictor
complex is abundant in both compartments. Rapamycin negatively regulates the
formation of both mTOR complexes, but the molecular mechanism of its effects on
mTORC2 remained elusive. We describe that in primary cells short-term treatment
with rapamycin triggers dephosphorylation of rictor and sin1 exclusively in the
cytoplasm, but does not affect mTORC2 assembly. Prolonged drug treatment leads
to complete dephosphorylation and cytoplasmic translocation of nuclear rictor
and sin1 accompanied by inhibition of mTORC2 assembly. The distinct cytoplasmic
and nuclear upstream and downstream effectors of mTOR are involved in many
cancers and human genetic diseases, such as tuberous sclerosis, Peutz-Jeghers
syndrome, von Hippel-Lindau disease, neurofibromatosis type 1, polycystic kidney
disease, Alzheimer's disease, cardiac hypertrophy, obesity and diabetes.
Accordingly, analogs of rapamycin are currently tested in many different
clinical trials. Our data allow new insights into the molecular consequences of
mTOR dysregulation under pathophysiological conditions and should help to
optimize rapamycin treatment of human diseases. The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and
PRAS40. Previously, we reported that mTOR plays an important role in regulating
protein synthesis in response to alcohol (EtOH). However, the mechanisms by
which EtOH regulates mTORC1 activity have not been established. Here, we
investigated the effect of EtOH on the phosphorylation and interaction of
components of mTORC1 in C2C12 myocytes. We also examined the specific role that
PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h)
increased raptor and PRAS40 phosphorylation. Likewise, there were increased
levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes
in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and
PRAS40 with mTOR. These alterations occurred in concert with increased binding
of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected.
The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to
EtOH. PRAS40 KD increased raptor phosphorylation and its association with
14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD
were mediated by AMPK. Both factors increased in vitro AMPK activity towards the
substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2.
Collectively, our results indicate that EtOH stabilizes the association of
raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction
of raptor with 14-3-3. These data suggest a possible mechanism for the
inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in
myocytes. The mTOR (mammalian target of rapamycin) protein kinase is an important
regulator of cell growth and is a key target for therapeutic intervention in
cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1),
consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8
(mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of
mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated
protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both
complexes phosphorylate the hydrophobic motifs of AGC kinase family members:
mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation
of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced
protein kinase 1). To investigate the roles of the Protor isoforms, we generated
single as well as double Protor-1- and Protor-2-knockout mice and studied how
activation of known mTORC2 substrates was affected. We observed that loss of
Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2
components, nor their ability to assemble into an active complex. Moreover,
Protor knockout mice display no defects in the phosphorylation of Akt and PKCα
at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1
knockout mice displayed markedly reduced hydrophobic motif phosphorylation of
SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the
kidney. Taken together, these results suggest that Protor-1 may play a role in
enabling mTORC2 to efficiently activate SGK1, at least in the kidney. LST8 is a component of both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).
Herein, to examine the role of LST8, a common component of mTOR complexes, in
the regulation of mTORC1 and mTORC2, first, we showed over-expression of LST8 in
HepG2 to markedly enhance basal phosphorylation levels of not only p70 S6 kinase
but also Akt. In contrast, LST8 knockdown by siRNA in HepG2 decreased
phosphorylation levels of both p70 S6 kinase and Akt. These results indicate the
LST8 expression level to determine basal mTORC1 and mTORC2 activities, since
LST8 appears to be the component present at the lowest level in both mTORC1 and
mTORC2 complexes. Previously, we reported S6 kinase phosphorylation to be
reduced by over-expression of the Cterminally deleted Raptor mutant (Raptor-ΔCT)
not binding to mTOR or LST8, while phosphorylation levels of Akt were markedly
enhanced with no alteration in IRS-1 phosphorylation or PI 3-kinase activity.
Using Raptor-ΔCT, we investigated the competition for association with LST8
between mTORC1 and mTORC2. Over-expression of Raptor-ΔCT abolished formation of
the Raptor, S6 kinase, mTOR and LST8 complex, while the amount of LST8 in the
Rictor-mTOR complex was increased. Therefore, it is likely that Raptor-mTOR and
Rictor-mTOR complexes compete for association with LST8, and this mechanism may
contribute to the reciprocal negative regulations of mTORC1 and mTORC2
activities, in terms of their LST8 components.: |
What is the the Menzerath-Altmann law? | Recently, a random breakage model has been proposed to explain the negative correlation between mean chromosome length and chromosome number that is found in many groups of species and is consistent with Menzerath-Altmann law, a statistical law that defines the dependency between the mean size of the whole and the number of parts in quantitative linguistics. The random breakage and variants keeping genome size and chromosome number independent raise no serious objection to the relevance of correlations consistent with Menzerath-Altmann law across taxonomic groups and the possibility of a connection between human language and genomes through that law. | Recently, a random breakage model has been proposed to explain the negative
correlation between mean chromosome length and chromosome number that is found
in many groups of species and is consistent with Menzerath-Altmann law, a
statistical law that defines the dependency between the mean size of the whole
and the number of parts in quantitative linguistics. Here, the central
assumption of the model, namely that genome size is independent from chromosome
number is reviewed. This assumption is shown to be unrealistic from the
perspective of chromosome structure and the statistical analysis of real
genomes. A general class of random models, including that random breakage model,
is analyzed. For any model within this class, a power law with an exponent of -1
is predicted for the expectation of the mean chromosome size as a function of
chromosome length, a functional dependency that is not supported by real
genomes. The random breakage and variants keeping genome size and chromosome
number independent raise no serious objection to the relevance of correlations
consistent with Menzerath-Altmann law across taxonomic groups and the
possibility of a connection between human language and genomes through that law. |
Which receptor(s) recognize lysosomal hydrolases in trans-Golgi network (TGN)? | The majority of lysosomal hydrolases in trans-Golgi network (TGN) are specifically recognized by mannose 6-phosphate (M6P) receptors (MPRs), which ensure their transport to the endosomal/lysosomal system. Other receptors can also carry lysosomal hydrolases from the trans-Golgi network (TGN) to the endosomal system. These M6P alternative receptors are the lysosomal integral membrane protein (LIMP-2) and the multi-ligand receptor sortilin. These reseptors enable a mannose-6-phosphate-independent pathway from TGN to lysosomes. | Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role
for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene,
in the targeting of newly synthesized proteins to the vacuole, an organelle
functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J.
Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC].
260:88-91). The activity of Vps34p kinase is significantly reduced by the PI
3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin
analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We
show here that at concentrations which inhibit VPS34-encoded PI 3-kinase
activity, wortmannin also inhibits the processing and delivery of newly
synthesized cathepsin D to lysosomes in mammalian cells with half-maximal
inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin
action, newly synthesized, unprocessed cathepsin D is secreted into the media.
Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C,
cathepsin D was rapidly missorted to the secretory pathway after addition of
wortmannin and shifting to 37 degrees C. At concentrations that inhibited
lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific
dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the
prelysosome compartment (PLC), which swelled to approximately 1 micron within 15
min after treatment. With increasing time, the inhibitors caused a significant
yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC
vacuoles were essentially depleted of receptors and, in addition, there was a
fourfold loss of receptors from the cell surface. However, M6PRs were still
abundant in the TGN. These results are most consistent with the interpretation
that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering
with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence
that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles
rely on similar regulatory mechanisms. At present little is known of the biochemical machinery controlling transport of
newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to
endosomes. The demonstration that Vps34p (a protein required for targeting
soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a
phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a
homologous enzyme might be involved in the equivalent step in mammalian cells.
Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to
support this hypothesis. Treatment of K-562 cells with wortmannin induced
secretion of procathepsin D, with half-maximal inhibition of accurate targeting
to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN)
step was affected, and that other constitutive vesicular transport events were
not. The M6P recognition signal was still generated in the presence of
wortmannin suggesting that the drug was directly inhibiting export of the
receptor-ligand complex from the TGN, while removal of the drug led to a rapid
restoration of accurate sorting. At the concentrations used, wortmannin and
LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude
that these data implicate such an enzyme in the trafficking of
M6P-receptor-ligand complexes from the TGN towards lysosomes. In many mammalian cells, the transport of newly synthesized or externally added
lysosomal enzymes to lysosomes is depend on their specific recognition by
receptors for mannose 6-phosphate (Man-6-P). The physiological importance of
this pathway was confirmed by the finding that fibroblasts from patients with
mucolipidosis type II (ML-II ; I - cell disease) fail to phosphorylate mannose
residues on their newly synthesized lysosomal enzymes, which results in the
secretion of a large percentage of their acid hydrolases into the culture
medium. However, lysosomal enzymes themselves do not contain the any consensus
amino acid sequences for acquiring the Man-6-P recognition marker. Kornfeld et
al revealed using cathepsin D-pepsinogen chimera proteins that
UDP-N-acetylglucosamine: lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase recognizes not only oligosaccharides
but also the three-dimensional structure of the lysosomal enzymes when transfers
N-acetylglucosamine-1-phosphate to lysosomal acid hydrolases. The localization and intracellular transport of major histocompatibility complex
(MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease
(ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent
targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC
class II-invariant chain complexes colocalized with the lysosomal hydrolase
cathepsin D in buds and vesicles that lacked markers of clathrin-coated
vesicle-mediated transport. These vesicles fused with the endocytic pathway
leading to the formation of "early" MHC class II-rich compartments (MIICs).
Similar structures were observed in the TGN of normal beta lymphoblasts although
they were less abundant. Metabolic labeling and subcellular fractionation
experiments indicated that newly synthesized cathepsin D and MHC class
II-invariant chain complexes enter a non-clathrin-coated vesicular structure
after their passage through the TGN and segregation from the secretory pathway.
These vesicles were also devoid of the cation-dependent mannose 6-phosphate
(Man-6-P) receptor, a marker of early and late endosomes. These findings suggest
that in ICD B lymphoblasts the majority of MHC class II molecules are
transported directly from the TGN to "early" MIICs and that acid hydrolases cam
be incorporated into MIICs simultaneously by a Man-6-P-independant process. Addition of wortmannin to normal rat kidney cells caused a redistribution of the
lysosomal type I integral membrane proteins Igp110 and Igp120 to a swollen
vacuolar compartment. This compartment did not contain the cation independent
mannose 6-phosphate receptor and was depleted in acid hydrolases. It was
distinct from another swollen vacuolar compartment containing the cation
independent mannose 6-phosphate receptor. The swollen Igp110-positive
compartment was accessible to a monoclonal antibody against Igp120 added
extracellularly, showing that it had the characteristics of an endosomal
compartment. Wortmannin had no gross morphological effect on the trans-Golgi
network or lysosomes nor any effect on the delivery to the trans-Golgi network
of endocytosed antibodies against the type I membrane protein TGN38. We propose
that the observed effects of wortmannin were due to inhibition of membrane
traffic between cation independent mannose 6-phosphate receptor-positive late
endosomes and the trans-Golgi network and to inhibition of membrane traffic
between a novel Igp120-positive, cation independent mannose 6-phosphate
receptor-negative late endosomal compartment and lysosomes. The effects of
wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in
regulating membrane traffic in the late endocytic pathway. Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from
the trans-Golgi network to endosomes, then return to the trans-Golgi network for
another round of enzyme delivery. Wortmannin, an inhibitor of
phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized
lysosomal enzymes to lysosomes. We used two independent assays of mannose
6-phosphate receptor trafficking to determine the precise step that is blocked
by wortmannin. Using an assay that monitors resialylation of desialylated cell
surface 300-kDa mannose 6-phosphate receptors, we found that receptor
endocytosis and transport to the trans-Golgi network were not inhibited by 2
microM wortmannin. In addition, this concentration of drug had no effect on the
transport of the mannose 6-phosphate receptor from late endosomes to the
trans-Golgi network using a system that reconstitutes this transport process in
cell extracts. Under the same conditions, wortmannin significantly inhibited the
generation of mature cathepsin D. In addition, the structurally unrelated
phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when
added to in vitro endosome-trans-Golgi network transport reactions. These
experiments demonstrate that the interruption in lysosomal enzyme targeting is
most likely due to a wortmannin-sensitive process required for the export of
these receptors from the trans-Golgi network, consistent with the established
role of phosphatidylinositol 3-kinase in the equivalent transport process in
Saccharomyces cerevisiae. A crucial step in lysosomal biogenesis is catalyzed by "uncovering" enzyme
(UCE), which removes a covering N-acetylglucosamine from the mannose 6-phosphate
(Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE
resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma
membrane. The cytosolic domain of UCE contains two potential endocytosis motifs:
(488)YHPL and C-terminal (511)NPFKD. YHPL is shown to be the more potent of the
two in retrieval of UCE from the plasma membrane. A green-fluorescent
protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN
46, as does endogenous UCE in HeLa cells, showing that the transmembrane and
cytosolic domains determine intracellular location. These data imply that the
Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P
receptors bind cargo and are packaged into clathrin-coated vesicles. The soluble hydrolases of the mammalian lysosome are marked for delivery to this
organelle by the addition of mannose 6-phosphate to their N-glycans. Two related
mannose 6-phosphate receptors (MPRs) recognize this feature in the trans Golgi
network (TGN) and deliver the hydrolases to the late endosome. In contrast, the
vacuolar hydrolases of the yeast Saccharomyces cerevisiae do not contain
6-phosphate monoesters on their N-glycans, and the only sorting receptor so far
identified in this organism is the product of the VPS10 gene. This protein also
cycles between the Golgi and the late endosome, but is unrelated to the
vertebrate MPRs, and recognizes a specific amino acid sequence of
carboxypeptidase Y (CPY). This has led to the notion that although yeast and
mammals share many components in Golgi to endosome traffic, they use unrelated
receptor systems to sort their abundant soluble hydrolases. In this paper, we
report that the yeast genome does in fact contain an uncharacterized ORF
(YPR079w) that encodes a membrane protein that is distantly related to mammalian
MPRs. The protein encoded by this gene (which we term MRL1) cycles through the
late endosome. Moreover, there is a strong synergistic effect on the maturation
of proteinases A and B when both MRL1 and VPS10 are deleted, which suggests that
Mrl1p may serve as a sorting receptor in the delivery of vacuolar hydrolases. The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of
lysosomal hydrolase precursors from the TGN to endosomes. After releasing the
hydrolase precursors into the endosomal lumen, the unoccupied receptor returns
to the TGN for further rounds of sorting. Here, we show that the mammalian
retromer complex participates in this retrieval pathway. The hVps35 subunit of
retromer interacts with the cytosolic domain of the CI-MPR. This interaction
probably occurs in an endosomal compartment, where most of the retromer is
localized. In particular, retromer is associated with tubular-vesicular profiles
that emanate from early endosomes or from intermediates in the maturation from
early to late endosomes. Depletion of retromer by RNA interference increases the
lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal
hydrolases, and causes swelling of lysosomes. These observations indicate that
retromer prevents the delivery of the CI-MPR to lysosomes, probably by
sequestration into endosome-derived tubules from where the receptor returns to
the TGN. The delivery of mannose 6-phosphate receptors carrying lysosomal hydrolases from
the trans-Golgi network (TGN) to the endosomal system is mediated by selective
incorporation of the receptor-hydrolase complexes into vesicular transport
carriers (TCs) that are coated with clathrin and the adaptor proteins, GGA and
AP-1. Previous electron microscopy (EM) and biochemical studies have shown that
these TCs consist of spherical coated vesicles with a diameter of 60-100 nm. The
use of fluorescent live cell imaging, however, has revealed that at least some
of this transport relies on a subset of apparently larger and highly
pleiomorphic carriers that detach from the TGN and translocate toward the
peripheral cytoplasm until they meet with distally located endosomes. The
ultrastructure of such long-range TCs has remained obscure because of the
inability to examine by conventional EM the morphological details of rapidly
moving organelles. The recent development of correlative light-EM has now
allowed us to obtain ultrastructural 'snapshots' of these TCs immediately after
their formation from the TGN in live cells. This approach has revealed that such
carriers range from typical 60- to 100-nm clathrin-coated vesicles to larger,
convoluted tubular-vesicular structures displaying several coated buds. We
propose that this subset of TCs serve as vehicles for long-range distribution of
biosynthetic or recycling cargo from the TGN to the peripheral endosomes. The sorting of acid hydrolase precursors at the trans-Golgi network (TGN) is
mediated by binding to mannose 6-phosphate receptors (MPRs) and subsequent
capture of the hydrolase-MPR complexes into clathrin-coated vesicles or
transport carriers (TCs) destined for delivery to endosomes. This capture
depends on the function of three monomeric clathrin adaptors named GGAs. The
GGAs comprise a C-terminal "ear" domain that binds a specific set of accessory
proteins. Herein we show that one of these accessory proteins, p56, colocalizes
and physically interacts with the three GGAs at the TGN. Moreover,
overexpression of the GGAs enhances the association of p56 with the TGN, and RNA
interference (RNAi)-mediated depletion of the GGAs decreases the TGN association
and total levels of p56. RNAi-mediated depletion of p56 or the GGAs causes
various degrees of missorting of the precursor of the acid hydrolase, cathepsin
D. In the case of p56 depletion, this missorting correlates with decreased
mobility of GGA-containing TCs. Transfection with an RNAi-resistant p56
construct, but not with a p56 construct lacking the GGA-ear-interacting motif,
restores the mobility of the TCs. We conclude that p56 tightly cooperates with
the GGAs in the sorting of cathepsin D to lysosomes, probably by enabling the
movement of GGA-containing TCs. The cation-independent mannose 6-phosphate receptor (CIMPR) cycles between the
trans-Golgi network (TGN) and endosomes to mediate sorting of lysosomal
hydrolases. The endosome-to-TGN retrieval of the CIMPR requires the retromer
complex. Genetic, biochemical and structural data support the hypothesis that
the retromer can directly bind to the tail of the CIMPR, to sort the CIMPR into
vesicles and tubules for retrieval to the TGN. Presently, however, no known
retromer sorting motif in the tail of the CIMPR has been identified. Using
CD8-reporter proteins carrying the cytoplasmic tail of the CIMPR we have
systematically dissected the CIMPR tail to identify a novel, conserved
aromatic-containing sorting motif that is critical for the endosome-to-TGN
retrieval of the CIMPR and for the interaction with retromer and the clathrin
adaptor AP-1. Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the
trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor
sortilin has also been implicated in this transport, but the transport carriers
involved herein have not been identified. By quantitative immuno-electron
microscopy, we localized endogenous sortilin of HepG2 cells predomitly to the
TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same
clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in
sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small
interfering RNA resulted in decreased pools of sortilin in the TGN and an
increase in lysosomal degradation. These data indicate that sortilin and MPRs
recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN
transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack
recycling plasma membrane proteins and by three-dimensional electron tomography
exhibit unique structural features. Hence, ETCs are distinct from hitherto
described EE-derived membranes involved in recycling. Our data emphasize an
important role of EEs in recycling to the TGN and indicate that different,
specialized exit events occur on the same EE vacuole. Three mammalian GGAs (Golgi-localized, gamma-ear-containing, ARF-binding
proteins), GGA1, 2, and 3 have been implicated in the sorting of mannose
6-phosphate receptor (MPR). To investigate the distinct roles of GGA2 in
lysosomal enzyme transport, we established two stable cell lines that had a
reduced expression of GGA2 by RNA interference. The expression levels of GGA2
were approximately 5% of the control levels, whereas those of non-targeted GGA1
and GGA3 were not apparently reduced. The depletion of GGA2 did not cause
changes in the overall distribution of GGA1, GGA3, cation-dependent MPR, or
cation-independent MPR. However, the cell lines showed increased secretion of a
lysosomal enzyme, cathepsin D. In addition, a moderate expression of the
domit negative VHS-GAT domain of GGA2 had no effect on the trans-Golgi
network (TGN) signal of three GGAs, nor was the GGA2 signal affected by the
expression of VHS-GAT domain of GGA1 or 3. These results suggest that GGA2 is
recruited to the TGN independently of the other GGAs and is required for the
efficient sorting of lysosomal enzymes. The delivery of soluble lysosomal proteins to the lysosomes is dependent
primarily on the mannose 6-phosphate receptor (MPR). The MPR has been
demonstrated to attain the early endosomes via a process that requires the
interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins.
Additionally, the MPR can be recycled back to the trans-Golgi network (TGN)
through its interaction with the retromer complex. Interestingly, in I-cell
disease (ICD), in which the MPR pathway is non-functional, many soluble
lysosomal proteins continue to traffic to the lysosomes. This observation led to
the discovery that sortilin is responsible for the MPR-independent targeting of
the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More
recently, our laboratory has tested the hypothesis that sortilin is also capable
of sorting a variety of cathepsins that exhibit varying degrees of
MPR-independent transport. We have demonstrated that the transport of cathepsin
D is partially dependent upon sortilin, that cathepsin H requires sortilin, and
that cathepsins K and L attain the lysosomes in a sortilin-independent fashion.
As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It
has been observed that like the MPR, the anterograde trafficking of sortilin and
its cargo require both GGAs and AP-1. Similarly, the retrograde recycling
pathway of sortilin also involves an interaction with retromer through a YXXphi
site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of
sortilin and MPR possess a high degree of functional homology and both receptors
share a conserved trafficking mechanism. Lysosomal hydrolases have long been known to be responsible for the degradation
of different substrates in the cell. These acid hydrolases are synthesized in
the rough endoplasmic reticulum and transported through the Golgi apparatus to
the trans-Golgi network (TGN). From there, they are delivered to
endosomal/lysosomal compartments, where they finally become active due to the
acidic pH characteristic of the lysosomal compartment. The majority of the
enzymes leave the TGN after modification with mannose-6-phosphate (M6P)
residues, which are specifically recognized by M6P receptors (MPRs), ensuring
their transport to the endosomal/lysosomal system. Although M6P receptors play a
major role in the intracellular transport of newly synthesized lysosomal enzymes
in mammalian cells, several lines of evidence suggest the existence of
alternative processes of lysosomal targeting. Among them, the two that are
mediated by the M6P alternative receptors, lysosomal integral membrane protein
(LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be
implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes,
whereas sortilin has been suggested to be a multifunctional receptor capable of
binding several different ligands, including neurotensin and receptor-associated
protein (RAP), and of targeting several proteins to the lysosome, including
sphingolipid activator proteins (prosaposin and GM2 activator protein), acid
sphingomyelinase and cathepsins D and H. Here, we review the current knowledge
on these two proteins: their discovery, study, structural features and cellular
function, with special attention to their role as alternative receptors to
lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to
disease are also extensively reviewed. |
List the diseases that can be treated using Vedolizumab. | Ulcerative colitis and Crohn's disease are inflammatory bowel diseases that have been successfully treated with Vedolizumab, a gut-selective, anti-inflammatory monoclonal antibody. | Inflammatory bowel diseases share common pathogenetic mechanisms that are not
yet completely understood. It is clear, however, that the expression and
production of cytokines in response to inflammation plays a key role in
mediating the migration of activated leukocytes. The process of angiogenesis and
the expression of adhesion molecules on the intestinal microvasculature act as
gateways, facilitating the recruitment of leukocytes into the gut mucosa. New
agents specifically blocking adhesion molecules, in particular integrins, have
been developed in order to limit the passage of activated leukocytes into the
mucosa. Non-gut-specific anti-integrin agents, such as natalizumab, have been
shown to be effective in the treatment of IBD, but the risk of serious adverse
events has limited their further development. The development of a new specific
molecule, vedolizumab, is currently under investigation in a large clinical
trial. This novel specific anti-integrin drug seems to hold promise in the
treatment of gut inflammation. Advances in immunology and genetics have identified new therapeutic targets to
control inflammation and symptoms in patients with inflammatory bowel diseases
(IBD). Despite the success of anti-TNF therapies in the treatment of IBD, a
considerable proportion of patients are refractory to treatment, highlighting an
unmet medical need for new therapies. Molecules that direct the trafficking of
inflammatory cells, such as the α4β7 integrin, are attractive targets for new
drug candidates. The α4β7 integrin is involved in lymphocyte recruitment to the
normal and inflamed gut mucosa, and the lymphoid tissue. The pan-α4 integrin
neutralizing mAb, natalizumab, is not gut-selective but has demonstrated
efficacy in IBD. However, treatment was associated with the occurrence of
progressive multifocal leukoencephalopathy, which has limited its use,
especially in Europe. Vedolizumab (MNL-0002), Millennium Pharmaceutical's
gut-specific, α4β7 integrin-neutralizing mAb, does not affect peripheral blood
cell counts and appears to lack systemic effects. Data from phase II clinical
trials of vedolizumab demonstrated efficacy with an attractive safety profile,
especially in ulcerative colitis. Large phase III, multicenter trials in both
ulcerative colitis and Crohn's disease will provide valuable data for the
ongoing development of vedolizumab, which might evolve as a new
anti-inflammatory treatment option for the management of therapy-refractory
patients. Inflammatory bowel diseases (IBD) are chronic relapsing and remitting disorders
that have varying degrees of severity. However multiple studies have confirmed
that a large proportion of patients on maintece treatment lose response to
anti-TNF therapy. This has led to increasing interest in the concept of
'switching therapy out-of-class' i.e. a noti- TNF antibody when patients
either fail to respond (primary non-response, develop secondary non-response) or
do not tolerate anti-TNF therapies. The most widely known and studied
alternative class of antibodies therapies at present are the selective adhesion
molecule inhibitors. Several antibodies exist which constitute selection
adhesion molecule inhibitors, including Natalizumab, MLN-0002 (Vedolizumab) and
ISIS 2302 (Alicaforsen) will be discussed in this review. The new information presented in Digestive Disease Week has allowed us to
speculate on the future of inflammatory bowel disease. Manipulation of diet and
the microbioma will probably play an increasingly important role in the
treatment of this disease and, in the long term, in its prevention. Biological
agents will probably be used earlier and more widely; new information on levels
of biological agents, mucosal healing and new comparative studies will also
allow these agents to be used in a more precise and personalized way. In
addition to infliximab, adalimumab, natalizumab and certolizumab, other
biological agents will be employed; among the first of these to be used will be
ustekinumab, golimumab and vedolizumab. In the near future, biological agents
will be used as frequently in ulcerative colitis as in Crohn's disease. New
healthcare models will be developed that will progressively include greater
participation among patients and nurses. The ability to predict new diagnostic
and prognostic models will allow decisions to be more individualized. Lymphocyte infiltration into the intestinal tract in inflammatory bowel disease
(IBD) is mediated by interaction between α4 integrin and its specific ligands.
Development of monoclonal antibodies against α4 integrin allowed targeting of
lymphocyte trafficking into the intestine as a novel therapeutic intervention.
Natalizumab, vedolizumab, alicaforsen AJM300, rhuMAb β7, CCX282-B, and
PF-00547,659 are few of monoclonal antibodies that have shown high promise in
trials with the potential for more attractive benefit:risk ratio than currently
available therapies. In this review, an attempt is made to underline the
therapeutic potential and the safety of anti-adhesion molecule treatment in IBD. INTRODUCTION: Crohn's disease (CD) is a chronic inflammatory disorder of unknown
aetiology. Currently, approved therapies that include prednisone,
anti-metabolites and TNF antagonists, are often ineffective and frequently cause
adverse effects. As a result, patients with CD can develop serious complications
that adversely affect quality of life. Consequently, new treatment options are
needed.
AREAS COVERED: This review discusses the potential role of vedolizumab, a
humanised monoclonal antibody that selectively blocks lymphocyte trafficking to
the gut, for the treatment of CD. All randomised placebo-controlled trials that
evaluated vedolizumab for the treatment of CD were reviewed and safety and
efficacy data evaluated.
EXPERT OPINION: Vedolizumab is an effective and well-tolerated drug that is an
important advance for the treatment of CD. Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases
that have been treated with corticosteroids, 5-aminosalicates and thiopurines,
but therapeutic options have been broadened with the arrival of anti-tumor
necrosis factor antibodies. In this article we reviewed the current
evidence-based approach to inflammatory bowel disease, the modifications that
have been made to existing therapies and discussed new drugs that have shown
success in clinical trials. The new drugs discussed here are those that disturb
lymphocyte homing to the gut (natalizumab, vedolizumab and anti-mucosal
addressin cellular adhesion molecule); one that blocks interleukin (IL)-12 as
well as the IL-23/T helper 17 (Th17) axis (ustekinumab) and one that blocks the
signaling of multiple cytokines (tofacitinib). Vedolizumab, a gut-homing α4β7 integrin antagonist, has demonstrated efficacy in
ulcerative colitis and Crohn's disease. Development of progressive multifocal
leukoencephalopathy, a serious brain infection associated with natalizumab (an
α4β7 and α4β1 integrin antagonist), has raised concern that vedolizumab may
convey a similar risk. Natalizumab is believed to impair central nervous system
immune surveillance by affecting cerebrospinal fluid (CSF) lymphocyte counts and
the CD4:CD8 ratio. To determine if vedolizumab elicits similar effects, we
examined CSF of healthy volunteers by flow cytometry for T-lymphocyte surface
markers 5 weeks after administration of intravenous vedolizumab 450 mg. No
significant changes were observed in CSF T-lymphocyte populations. The presentations at Digestive Disease Week 2013 emphasized treatment safety.
Anti-tumor necrosis factor (TNF) agents and thiopurines are reasonably safe in
breastfeeding and pregcy. Several studies indicate that controlling the risk
of tuberculosis when anti-TNF agents are planned presents several problems, both
in the initial diagnosis of latent tuberculosis and in subsequent patient
follow-up, given that cases of tuberculosis continue to occur, despite
recommendations. Thiopurines increase the risk of lymphoma, but there is no
residual risk when these drugs are withdrawn. Despite increasing knowledge of
the risks and recommendations on how to avoid them, there remain considerable
shortfalls in the application of preventive measures and, more specifically, in
vaccinations. Infliximab and cyclosporin produce similar results when used to
treat severe outbreaks of ulcerative colitis. Thromboembolism prevention
continues to be deficient, and the barriers to effective prevention concern not
only physicians but can also involve nursing staff, for example. There is still
a wide margin for improvement in safety. New drugs under study (vedolizumab,
golimumab) have not shown any hitherto unknown signs of significant toxicity. Several studies on conventional drugs and new treatments in inflammatory bowel
disease were presented in Digestive Disease Week 2013. Various studies have
compared infliximab and cyclosporin in corticosteroid-refractory ulcerative
colitis in clinical practice, providing complementary information to the CYSIF
clinical trial. For the first time, a clinical trial has evaluated the efficacy
of adalimumab in preventing recurrence of Crohn's disease after surgery. The
results of some studies suggest that thiopurines improve response to infliximab,
in both Crohn disease and ulcerative colitis. Finally, several studies were
presented on new drugs with new therapeutic targets, such as vedolizumab, in the
treatment of inflammatory bowel disease. The preliminary results of the ASTIC
trial were reported, which evaluated the safety and efficacy of bone marrow
transplantation in Crohn's disease. During the open-label trial of natalizumab for Crohn's disease, an isolated case
of progressive multifocal leukoencephalopathy (PML) was found. This prompted a
more careful review by regulators, physicians, and the pharmaceutical industry.
A new gut-specific monoclonal antibody, vedolizumab, has been shown to be
effective in inflammatory bowel disease, and in continued trials no patients
have developed PML. Given the mortality of PML and lack of effective treatments,
patients may remain concerned that PML is a possible risk factor. So, going
forward, how do we quantify the risk of this serious adverse event? This review
details how we define the maximum risk when no (or very few) events have
occurred with an easy-to-use equation. The causes of inflammatory bowel diseases, such as ulcerative colitis (UC) and
Crohn's disease (CD), remain to be elucidated. However, characteristic
inflammation of the gastrointestinal mucosa is caused by infiltration of T
lymphocytes into the submucosal layer. Inhibiting this immune response is a
promising therapeutic target. Integrins expressed on the cell surface mediate
gut homing of T lymphocytes. Blockade of integrin-cell adhesion molecule
interaction using antibodies against α₄-containing integrins, namely
natalizumab, has shown clinical efficacy; however, this drug's lack of
α₄-containing integrin specificity leads to systemic immunosuppression that
caused progressive multifocal leukoencephalopathy and death in some patients
resulting in its withdrawal from the market. Vedolizumab specifically targets
the α₄β₇ integrin that is selectively expressed on gut-homing T lymphocytes.
Vedolizumab successfully extended clinical remission in patients with UC or CD
and reduced patient reliance on corticosteroid use. The drug is well tolerated
and there have been no deaths or reports of progressive multifocal
leukoencephalopathy infection in patients receiving vedolizumab. A phase III
long-term 7-year safety study in patients with UC and CD is under way.
Regulatory applications are under review in the U.S. and E.U. for its use in the
treatment of patients with UC and CD, with decisions expected in mid-2014. Vedolizumab [Entyvio(®) (US, Europe)], a humanized monoclonal antibody α4β7
integrin receptor antagonist, has been developed by Millennium Pharmaceuticals
(d/b/a Takeda Pharmaceuticals International) for the treatment of ulcerative
colitis and Crohn's disease. Vedolizumab has received its first global approval
for the treatment of ulcerative colitis and Crohn's disease in the US, for use
in adult patients with moderate-to-severe disease who have had an inadequate
response, loss of response or intolerance to one or more standard therapies
(corticosteroids, immunomodulators or tumour necrosis factor-α inhibitor) or
demonstrated dependence on corticosteroids. Vedolizumab has since been approved
for ulcerative colitis and Crohn's disease in the EU, Norway, Iceland and
Liechtenstein. This article summarizes the milestones in the development of
vedolizumab leading to its first approval for the treatment of ulcerative
colitis and Crohn's disease. BACKGROUND: Cellular adhesion molecules play an important role in the
pathogenesis of ulcerative colitis, making selective blockade of these molecules
a promising therapeutic strategy. Vedolizumab, a recombit humanized IgG1
monoclonal antibody, inhibits adhesion and migration of leukocytes into the
gastrointestinal tract by binding the alpha4beta7 integrin. Animal studies have
suggested that vedolizumab may be a useful therapy for ulcerative colitis. This
updated systematic review summarizes the current evidence on the use of
vedolizumab for induction and maintece of remission in ulcerative colitis.
OBJECTIVES: The primary objectives were to determine the efficacy and safety of
vedolizumab used for induction and maintece of remission in ulcerative
colitis.
SEARCH METHODS: A computer-assisted search for relevant studies (inception to 15
June 2014) was performed using PubMed, MEDLINE, EMBASE and CENTRAL. References
from published articles and conference proceedings were searched to identify
additional citations.
SELECTION CRITERIA: Randomized controlled trials comparing vedolizumab to
placebo or a control therapy for induction or maintece of remission in
ulcerative colitis were included.
DATA COLLECTION AND ANALYSIS: Two authors independently extracted data and
assessed the risk of bias for each trial. The primary outcomes were failure to
induce clinical remission and relapse. Secondary outcomes included failure to
induce a clinical response, failure to induce endoscopic remission, failure to
induce an endoscopic response, quality of life, adverse events, serious adverse
events and withdrawal due to adverse events. We calculated the relative risk
(RR) and 95% confidence intervals (CI) for each outcome. Data were analyzed on
an intention-to-treat basis. The overall quality of the evidence supporting the
outcomes was evaluated using the GRADE criteria.
MAIN RESULTS: Four studies (606 patients) were included. All of the studies were
rated as having a low risk of bias. Pooled analyses revealed that vedolizumab
was significantly superior to placebo for induction of remission, clinical
response, and endoscopic remission and prevention of relapse. After 4 to 6 weeks
of therapy 77% (293/382) of vedolizumab patients failed to enter clinical
remission compared to 92% (205/224) of placebo patients (RR 0.86, 95% CI 0.80 to
0.91; 4 studies 606 patients). After 6 weeks of therapy 48% of vedolizumab
patients failed to have a clinical response compared to 72% of placebo patients
(RR 0.68, 95% CI 0.59 to 0.78; 3 studies 601 patients). After 4 to 6 weeks of
therapy 68% of vedolizumab patients failed to enter endoscopic remission
compared to 81% of placebo patients (RR 0.82, 95% CI 0.75 to 0.91; 3 studies,
b583 patients). After 52 weeks of therapy, 54% of vedolizumab patients had a
clinical relapse compared to 84% of placebo patients (RR 0.67, 95% CI 0.59 to
0.77; 1 study, 373 patients). One small study (28 patients) found no
statistically significant difference in endoscopic response (RR 1.00, 95% CI
0.62 to 1.61). GRADE analyses indicated that the overall quality of the evidence
for the primary outcomes was high for induction of remission and moderate for
relapse (due to sparse data 246 events). There was no statistically significant
difference between vedolizumab and placebo in terms of the risk of any adverse
event (RR 0.99, 95% CI 0.93 to 1.07), or serious adverse events (RR 1.01, 95% CI
0.72 to 1.42). There was a statistically significant difference in withdrawals
due to adverse events. Six per cent of vedolizumab patients withdrew due to an
adverse event compared to 11% of placebo patients (RR 0.55, 95% CI 0.35 to 0.87;
2 studies, 941 patients). Adverse events commonly reported across the studies
included: worsening ulcerative colitis, headache, nasopharyngitis, upper
respiratory tract infection, nausea, and abdominal pain.
AUTHORS' CONCLUSIONS: Moderate to high quality data from four studies shows that
vedolizumab is superior to placebo for induction of clinical remission and
response and endoscopic remission in patients with moderate to severely active
ulcerative colitis and prevention of relapse in patients with quiescent
ulcerative colitis. Moderate quality data from one study suggests that
vedolizumab is superior to placebo for prevention of relapse in patients with
quiescent ulcerative colitis. Adverse events appear to be similar to placebo.
Future trials are needed to define the optimal dose, frequency of administration
and long-term efficacy and safety of vedolizumab used for induction and
maintece therapy of ulcerative colitis. Vedolizumab should be compared to
other currently approved therapies for ulcerative colitis in these trials. Lymphocyte homing antagonists represent promising therapeutic agents for the
treatment of idiopathic inflammatory bowel disease (IBD). Several critical
molecules involved in the recruitment of inflammatory cells in the intestine,
including integrins and chemokine receptors, have been successfully targeted for
the treatment of IBD. These agents have shown great promise for the induction
and maintece of remission for both Crohn disease and ulcerative colitis. This
article discusses currently approved prototypic agents for the treatment of IBD
(natalizumab, anti-α4 integrin; vedolizumab, anti-α4β7 integrin), and several
other agents in the same class currently under development. OBJECTIVES: To review the pharmacology, efficacy, and safety of vedolizumab in
the treatment of patients with ulcerative colitis (UC) and Crohn's disease (CD).
DATA SOURCES: A literature search through clinicialtrials.gov, EMBASE and
MEDLINE was conducted (January 1966-June 2014) using the terms vedolizumab and
MLN0002. References from retrieved articles were reviewed for any additional
material. Additionally, the prescribing information was retrieved.
STUDY SELECTION/DATA EXTRACTION: Phase 1, 2, and 3 human and animal studies
describing the pharmacology, pharmacokinetics, efficacy, and safety of
vedolizumab were identified.
DATA SYNTHESIS: Vedolizumab, an α4β7 integrin inhibitor, was recently approved
for adult patients with moderate to severe active UC or CD who are refractory or
intolerant to standard therapies or who are dependent on corticosteroids. Trial
data have demonstrated that vedolizumab 300 mg at weeks 0, 2, and 6 followed by
every 8 weeks is effective at inducing and maintaining clinical response and
remission, improving mucosal appearance, and achieving corticosteroid-free
remission in patients with UC. This regimen is also effective at achieving
clinical response, remission, and corticosteroid-free remission in patients with
CD. Patients treated with vedolizumab, unadjusted for exposure, reported
experiencing nasopharyngitis, headache, nausea, arthralgias, pyrexia,
upper-respiratory-tract infections, fatigue, and cough.
CONCLUSIONS: Vedolizumab is an effective agent at inducing and maintaining
remission in patients with UC or CD. Vedolizumab is generally well tolerated and
has not been associated with progressive multifocal leukoencephalopathy. Two decades ago, the first reports of the use of monoclonal antibodies targeting
tumour-necrosis factor α heralded a revolution in treatment options for moderate
to severe Crohn's disease and ulcerative colitis. Nonetheless, patients with
refractory disease or loss of treatment response are all too familiar to
gastroenterologists. Preventing the infiltration of the gastrointestinal mucosa
by circulating cells of the immune system using antibodies targeting the
adhesion molecules involved represents an attractive new treatment option.
Vedolizumab has recently received European and US regulatory approval for
treatment of ulcerative colitis and Crohn's disease on the basis of encouraging
results from one of the largest phase III trial programmes ever conducted in the
field of inflammatory bowel diseases and promising safety data. Are we now
seeing another revolution in the management of inflammatory bowel disease, and
how can this new drug best be used in clinical practice? Vedolizumab (Entyvio™) is a humanized monoclonal antibody α4β7 integrin-receptor
antagonist indicated for the treatment of adult patients with moderately to
severely active ulcerative colitis or Crohn's disease. This article reviews the
pharmacological properties of intravenous infusions of vedolizumab and its
clinical efficacy in adult patients with these diseases. In phase III clinical
trials, patients with ulcerative colitis had significantly higher rates of
clinical response and clinical remission when treated with vedolizumab than when
receiving placebo at both 6 and 52 weeks. However, outcomes with vedolizumab in
patients with Crohn's disease were mixed. In a study that evaluated both
clinical remission rate and CDAI-100 response rate as primary endpoints, only
the clinical remission rate at 6 weeks was significantly higher with vedolizumab
than placebo. In another trial, there was no significant between-group
difference in the clinical remission rate in TNF-antagonist failure patients at
6 weeks (primary endpoint), although there was a significant difference at
10 weeks. In the Crohn's disease study that included maintece treatment,
vedolizumab was significantly more effective at 52 weeks than placebo in both
endpoints (clinical remission was the only primary endpoint in the maintece
study). Vedolizumab was generally well tolerated in these trials. As vedolizumab
is a specific α4β7 integrin antagonist, with gut-specific effects, it is
unlikely to be associated with the development of progressive multifocal
leukoencephalopathy, a risk observed with the less selective α4β7/α4β1 integrin
antagonist natalizumab. Vedolizumab is a useful addition to the treatment
options available for patients with moderately to severely active ulcerative
colitis and Crohn's disease. |
Can clonidine be used to reduce agitation in children. | Yes, clonidine is effective in prevention of post-anesthesia agitation in children. | In a double-blinded trial, 40 male children (age 2-7 yr) undergoing circumcision
were randomly assigned to receive clonidine 2 microg/kg IV or placebo after
anesthetic induction. For induction and maintece of anesthesia, we used
sevoflurane as the sole anesthetic. For pain treatment, a penile block was
performed before surgery. After surgery the incidence and severity of agitation
was measured during an observation period of 2 h. Severe agitation was treated
with midazolam. In 16 placebo and 2 clonidine-treated patients agitation was
observed (P < 0.001). In 6 patients of the Placebo group, agitation was graded
as severe, whereas none of the patients in the Clonidine group developed severe
agitation (P = 0.02). During the postoperative period heart rate and blood
pressure were significantly decreased in clonidine treated patients (P < 0.05).
We conclude that clonidine effectively prevents agitation after sevoflurane
anesthesia.
IMPLICATIONS: The recovery from sevoflurane anesthesia may be complicated by the
presence of agitation in pediatric patients. Clonidine 2 microg/kg IV after
anesthetic induction effectively reduces the incidence of agitation without
resulting in clinically relevant bradycardia and hypotension. BACKGROUND: This double-blind randomized study was undertaken to assess
agitation, Bispectral Index (BIS) and EEG changes during induction of
anaesthesia with sevoflurane in children premedicated with midazolam or
clonidine.
METHODS: Children were allocated randomly to receive rectal midazolam 0.4 mg
kg(-1) (n=20) or oral clonidine 4 microg kg(-1) (n=20) as premedication. Rapid
induction of anaesthesia was achieved with inhalation of sevoflurane 8% in
nitrous oxide 50%-oxygen 50%. After tracheal intubation, the children's lungs
were mechanically ventilated and the inspired sevoflurane concentration was
adjusted to achieve an end-tidal fraction of 2.5%. The EEG and BIS were recorded
during induction until 10 min after tracheal intubation. The EEG was analysed
using spectral analysis at five points: baseline, loss of eyelash reflex, 15 s
before the nadir of the BIS (BIS(nadir)), when both pupils returned to the
central position (immediately before intubation), and 10 min after intubation.
RESULTS: Agitation was observed in 12 midazolam-treated and five
clonidine-treated patients (P=0.05). At baseline, EEG rhythms were slower in the
clonidine group. Induction of anaesthesia was associated with similar EEG
changes in the two groups, with an increase in total spectral power and a shift
towards low frequencies; these changes were maximal around the end of the second
minute of induction (BIS(nadir)). When the pupils had returned to the central
position, fast EEG rhythms increased and BIS was higher than BIS(nadir)
(P<0.05). In both groups, agitation was associated with an increase in slow EEG
rhythms at BIS(nadir).
CONCLUSIONS: Compared with midazolam, clonidine premedication reduced agitation
during sevoflurane induction. During induction with sevoflurane 8% (oxygen
50%-nitrous oxide 50%), the nadir of the BIS occurred at the end of the second
minute of inhalation. Agitation was associated with a more pronounced slowing of
the EEG rhythms at BIS(nadir) compared with inductions in which no agitation was
observed. The BIS may not follow the depth of anaesthesia during sevoflurane
induction in children. Clonidine is effective in treating sevoflurane-induced postanesthesia agitation
in children. We conducted a study on 169 children to quantify the risk reduction
of clonidine agitation in patients admitted to our day-surgery pediatric clinic.
Children were randomly allocated to receive clonidine 2 mug/kg or placebo before
general anesthesia with sevoflurane that was also supplemented with a regional
or central block. An observer blinded to the anesthetic technique assessed
recovery variables and the presence of agitation. Pain and discomfort scores
were significantly decreased in the clonidine group; the incidence of agitation
was reduced by 57% (P = 0.029) and the incidence of severe agitation by 67% (P =
0.064). Relative risks for developing agitation and severe agitation were 0.43
(95% confidence interval, 0.24-0.78) and 0.32 (0.09-1.17), respectively.
Clonidine produces a substantial reduction in the risk of postsevoflurane
agitation in children. BACKGROUND: Emergence agitation (EA) is a common postoperative problem in young
children who have received sevoflurane and isoflurane for general anesthesia.
This randomized, double-blinded study evaluated the efficacy of intraoperative
clonidine in reducing EA, and describes its recovery profile.
METHODS: With Institutional Review Board approval and informed consent, children
undergoing brief, minimally painful procedures were studied. All children
received preemptive analgesia with acetaminophen and ketorolac, sevoflurane for
induction, and isoflurane for maintece of anesthesia. Children received
either 2 microg.kg(-1) clonidine or placebo intravenously (i.v.) following
induction of anesthesia. Children were observed postoperatively for behavior and
side effects, and their parents were telephoned the next day to determine
postdischarge recovery characteristics.
RESULTS: One hundred and twenty children were included in this study: 59 of whom
received clonidine, and 61 placebo; 41% of those in the placebo group exhibited
moderate-severe EA compared with only 22% of those in the clonidine group (P <
0.03). Compared with those who received placebo, children who received clonidine
awakened more slowly (22 min vs 14 min), had a longer postanesthesia care unit
stay (57 min vs 46 min), and experienced sleepiness more frequently after
discharge (75% vs 39%; all comparisons significant at P < 0.03). There were no
adverse cardiorespiratory events in either group.
CONCLUSIONS: Findings demonstrate that i.v. clonidine administered after
induction of anesthesia significantly reduces the incidence of EA in young
children, but is associated with sleepiness postoperatively. BACKGROUND: Oral premedication is widely used in pediatric anesthesia to reduce
preoperative anxiety and ensure smooth induction. Midazolam is currently the
most commonly used premedicant, but good results have also been reported with
clonidine. The aim of the present study was to compare clinical effects of oral
midazolam and oral clonidine.
METHODS: We performed a prospective open study in 64 children who were randomly
assigned to receive either oral midazolam 0.5 mg.kg (-1) (group M) or oral
clonidine 4 microg.kg (-1) (group C) prior to mask induction. Drug acceptance,
preoperative sedation and anxiolysis, quality of mask acceptance, recovery
profile and parental satisfaction were evaluated.
RESULTS: The taste of oral clonidine was judged as significantly better; 14% of
children rejected oral midazolam. Onset of sedation was significantly faster
after premedication with midazolam (30+/-13.1 min) than with clonidine
(38.5+/-14.6 min), but level of sedation was significantly better after
premedication with clonidine. Quality of mask induction was equally successful
in both groups. A steal-induction was performed in 66% of patients of group C,
but none in group M. We observed a trend towards an increased incidence of
emergence agitation after premedication with midazolam. Parental satisfaction
was significantly higher in group C.
CONCLUSIONS: In this study, premedication with oral clonidine appeared to be
superior to oral midazolam. Quality of mask acceptance was comparable between
groups, but oral clonidine was better accepted by the child, produced more
effective preoperative sedation, showed a trend towards better recovery from
anesthesia and had a higher degree of parental satisfaction. Clonidine is experiencing increasing use in the pediatric population as a
sedative and analgesic because of its central alpha2-adrenergic agonism. We
report three cases of preoperative use of intranasal clonidine in pediatric
patients, all for different indications. One patient was treated for
preoperative agitation and hallucinations associated with oral midazolam. One
patient was given clonidine as a premedicant. The third patient was treated for
preoperative agitation and hypertension. All three patients had subjective
resolution of indicated symptoms and none experienced adverse outcomes. This trial assessed the effects of two doses of clonidine compared with placebo
on the quality and speed of recovery in children premedicated with oral
midazolam and anaesthetised with sevoflurane for cataract surgery. One hundred
and twenty American Society of Anesthesiologists physical status I to II
children (aged one to six years), premedicated with oral midazolam 0.5 mg/kg and
undergoing elective unilateral cataract surgery with sevoflurane anaesthesia
were studied. Children were randomised to intravenous clonidine 1 microg/kg
(group C1, n=39), 2 microg/kg (group C2, n=41) or normal saline (group NS,
n=40). Clinically successful sub-Tenon local anaesthesia block was required for
a patient to be included in the analysis. The primary outcome was the incidence
of postoperative agitation. Postoperative agitation was defined as a Pain
Discomfort Score of -3 using items 3 to 5 only, which was assessed 15 minutely
until discharge. Agitation was observed in 11/40 (27.5%) children in the NS
group compared to 2/39 (5.1%) in group Cl and none in group C2 (P < 0.001).
Rescue medication to treat severe agitation was required in 5/40 (12.5%) in the
NS group, 1/39 (2.6%) in group C1 and none in group C2 (P = 0.025). Time to meet
discharge criteria was significantly shorter in group C1 compared to the other
two groups (48.4 +/- 14.0 minutes compared to C2 79.5 +/- 12.8 minutes and NS
73.1 +/- 20.4 minutes, P < 0.001). There were no significant effects on blood
pressure and heart rate. Intravenous clonidine 1 microg/kg is effective for
reducing agitation after sevoflurane anaesthesia and midazolam premedication in
children undergoing cataract surgery. Intravenous clonidine 2 microg/kg was also
effective and for a longer period, but was associated with a longer time to
discharge. BACKGROUND: Sevoflurane is commonly used as an inhalational induction agent in
paediatric patients. Emergence agitation is a common post-operative problem in
young children who have received sevoflurane. Clonidine has proven to be
effective in reducing the incidence of post-operative agitation at a higher dose
(3 and 2 μg kg⁻¹). It has some dose-dependent disadvantages, prominently
bradycardia, hypotension and respiratory impairment.
OBJECTIVE: The authors conducted a study to evaluate the effectiveness of
low-dose caudal clonidine (1 μg kg⁻¹) in reducing the incidence of
sevoflurane-induced agitation in preschool children undergoing urogenital and
lower limb surgery.
METHODOLOGY: A double-blind study was conducted comparing 0.25% (0.75 ml kg⁻¹)
bupivacaine and clonidine 1 μg kg⁻¹ (group 1), 0.25% bupivacaine (0.75 ml kg⁻¹)
and clonidine 0.75 μg kg⁻¹ (group 2), with 0.25% bupivacaine (0.75 ml kg⁻¹)
alone (group 3). Ninety children of 1-5 years of American Society of
Anesthesiologists I and II were randomly assigned into three groups.
Post-operatively, patients were monitored for 1 h to observe emergence
agitation, which was assessed with the help of Pain and Discomfort Scale.
RESULT: Post-anaesthetic agitation was observed in two patients (6.6%) in group
1, eight patients (26.6%) in group 2 as compared to 12 patients (40%) in group 3
after 15 min of post-operative observation. The mean scores in group 1 at 15 and
30 min were significantly lower than those in group 3 (P value <0.05). None of
the groups had showed any haemodynamic and respiratory compromise, either
clinically and statistically.
CONCLUSION: Caudal clonidine at a lower dose (1 μg kg⁻¹) could be effective in
reducing the incidence of sevoflurane-induced emergence agitation in children
undergoing urogenital and lower limb surgery without any significant adverse
effects. PURPOSE: Postoperative agitation is common in adults and children following the
use of several anesthetics, particularly inhalation anesthetics. This behavior
has detrimental effects both physically for the patient following the procedure
and psychologically for the parent or guardian. The authors propose that
clonidine, an alpha-2 agonist, would provide a reduction in children's
postoperative agitation and, in turn, improve parent satisfaction in children
undergoing strabismus surgery.
METHODS: Fifty children were prospectively randomized to receive either
clonidine or placebo in a double-blind fashion prior to surgery. The children
were evaluated using preoperative and postoperative parent questionnaires and
postoperative behavioral evaluation using standardized scales including the
Pediatric Anesthesia Emergence Delirium and Watcha scales.
RESULTS: Children receiving clonidine immediately after anesthesia induction had
statistically significant improvement in postoperative agitation at the
15-minute mark (P = .096) and last score obtained (P = .095) using the Watcha
scale. The clonidine group spent an insignificantly longer time in the
post-anesthesia care unit (average: 54 minutes) than the placebo group (average
47 minutes) (P = .17). Seventy-one percent of parents reported being "very
satisfied" with their child's recovery from anesthesia in the clonidine group
compared to 46% in the placebo group, although this was not statistically
significant (P = .34).
CONCLUSIONS: Children receiving clonidine prior to undergoing strabismus surgery
have a small but noticeable reduction in postoperative agitation, stay slightly
longer in the post-anesthesia care unit, and have higher rates of parent
satisfaction. |
Which value of nuchal translucency thickness is set as the threshold for high-risk for Down Syndrome? | NT is physiological for a measurement < 3 mm but the incidence of chromosomal abnormalities (essentially trisomies 21, 18 and 13) increases when NT > or = 3 mm. As women aged, this upper NT threshold value changed according to gestational age. In women aged 35 to 37 years, combined prenatal screening was always positive when NT exceeded 2.8 mm, 3.0 mm, and 3.4 mm at 11, 12, and 13 weeks of gestation, respectively. | The purpose of the present literature review is to assess the screening value of
trisomy 21 by measurement of fetal nuchal translucency (NT) thickness in the
first trimester. NT is a subcutaneous translucency between the skin and the soft
tissues overlying the cervical spine, which disappears in the second trimester.
Ultrasound examination was used to image a sagittal section of the fetus to
measure the maximum thickness of the subcutaneous translucency. NT is
physiological for a measurement < 3 mm but the incidence of chromosomal
abnormalities (essentially trisomies 21, 18 and 13) increases when NT > or = 3
mm. Differential diagnoses include cystic hygroma and fetal hydrops. For
screening purposes, a cut-off threshold value of > or = 3 mm, with a
standardized technique, gave a sensitivity > or = 50%, a false positive rate <
5% and a positive predictive value > 1%. In the chromosomally normal group,
prognosis was good, but incidence of structural defects and fetal loss
increased, with a sharp rise in these complications for fetal translucency
thickness > or = 5 mm. OBJECTIVES: The purpose of this study is to assess the feasibility of foetal
nasal bone (NB) measurement during the first trimester of pregcy, and to
examine the contribution of this measurement to the prenatal screening for Down
syndrome following the definition of NB threshold using ROC curves in an
unselected population.
METHODS: This prospective study was carried out at our centre SIHCUS-CMCO
(reference centre) from January 2002 to December 2004 on a total of 2,044
pregt outpatients at gestational weeks 11-14. Only 1260 singleton foetuses
were used for statistical analysis. In the 784 other patients, we were unable to
obtain a correct image allowing a reproducible measurement. NB was measured
during the same session as nuchal translucency (NT) measurement. Ten trained
sonographers took part in the study. Correlation index was evaluated to shed
light on a link between interest variables and NB. Screening values of NB
measurement in T 21 were also calculated with NB measurement according to
crown-rump length, and expressed as the best threshold of multiple of the median
determined by ROC curve. Screening values of genetic ultrasound were then
evaluated by adding NB measurement to maternal age and NT measurement.
RESULTS: Two thousand and forty-four patients were included. We indexed 30 cases
of T 21, 14 cases of Trisomy 18, 10 cases of Trisomy 13 and 25 cases of other
karyotype abnormalities. Feasibility of measurement was 62% of all cases. We
observed a significant relation between NB and NT (p = 0.001 ), as well as
between NB and crown-rump-length (p < 0.0001 ). However, size of NB was not
correlated to maternal ethnic group (p = 0.314). At 0.6 multiple of the median
thresholds, screening values of NB measurement in T 21 were: sensibility 32%,
false positive rate 10%, positive predictive value 13.6%, and negative
predictive value 96.9%. The likelihood ratio for T 21 in case of NB < or = 0.6
multiple of the median was 4.4 (2.0-9.4). Screening values for maternal age and
NT measurement were: sensitivity 88%, false positive rate 23%,positive
predictive value 9.7%, and negative predictive value 99.6%. Inclusion of NB
measurement increased sensitivity to 100%, positive predictive value to 13.6%,
and negative predictive value to 100%, and decreased false positive rate to 5%.
CONCLUSION: NB measurement seemed to be a great sonographic marker for T 21.
However, its low feasibility made it inadequate for routine settings in first
trimester T 21 screening in an unselected population. Statistical independence
with NT thickness needed to be further evaluated. OBJECTIVE: To determine if nuchal translucency (NT) can be used as a first
trimester triage marker in prenatal screening for Down syndrome and trisomy 18.
METHODS: Data from first trimester prenatal screening in 77 443 women were
stratified by maternal and gestational ages. They were then analyzed to identify
NT thresholds above or below which only positive (high-risk) or negative
(low-risk) results were reported by a first trimester prenatal screening test
combining PAPP-A, free beta-hCG and NT.
RESULTS: Combined prenatal screening was always positive for Down syndrome when
NT thickness exceeded 4.0 mm. As women aged, this upper NT threshold value
changed according to gestational age. In women aged 35 to 37 years, combined
prenatal screening was always positive when NT exceeded 2.8 mm, 3.0 mm, and 3.4
mm at 11, 12, and 13 weeks of gestation, respectively. In women over 42 years of
age, the upper threshold value for NT was 1.8 mm, 2.4 mm, and 2.7 mm at 11, 12,
and 13 weeks of gestation, respectively. In women less than 35 years of age, we
identified lower threshold values below which combined prenatal screening for
Down syndrome was always negative.
CONCLUSION: In prenatal screening for Down syndrome and trisomy 18, it is
possible to identify NT threshold values above which biochemical screening
provides no additional benefit. In pregcies in which NT is above the
established upper cut-offs, invasive prenatal screening can be offered without
delay. |
Does ventriculoperitoneal shunt improve normal pressure hydrocephalus? | Yes | A prospective trial for diagnosis, treatment and evaluation of normal pressure
hydrocephalus (NPH) was carried out in 30 consecutive patients (12 women, 18
men, mean age: 61 years, range 21-80). The clinical diagnosis was based on the
presence of one or more of the three clinical elements of NPH: gait disturbance,
incontinence and mental deterioration as well as radiological criteria of
ventricular enlargement. Preoperatively, physical and radiological
investigations and psychological tests were routinely performed. Cognitive
functions such as fluency, selective attention and motor speed were re-examined
within 24 hours after spinal tap of 40-50 cc. of CSF. Results of shunting were
confirmed by CT scans and evaluated by means of psychological tests at least 3
months postoperatively. Psychological tests included IQ tests and tests for
memory, selective attention and motor speed. Results indicate that spinal tap
had no predictive value for the results after shunting as far as psychological
functions are concerned. Shunt treatment showed an effect on cognitive functions
of distractibility of attention and motor speed, but not on intelligence of
memory. Three patients deteriorated, eleven remained stable and sixteen showed
significant improvement on psychological tests, mainly those for attention,
motor speed and memory, but rarely did any improvement of intelligence occur. Idiopathic normal pressure hydrocephalus (INPH) is a treatable neurological
disorder in older adults involving disturbances of gait/balance, control of
micturition, and/or cognition in combination with enlargement of the cerebral
ventricles. Diagnosis can be challenging due to its varied presentation and
overlap with other disorders common in the elderly. Evidence-based consensus
guidelines for diagnosis and treatment of INPH have been created that can assist
in clinical management. Diagnosis requires clinical documentation of one or more
of the characteristic symptoms of INPH in combination with a brain imaging study
demonstrating nonobstructive ventricular enlargement disproportionate to
cerebral atrophy. Gait and balance disturbances are the most common presenting
findings in INPH and may occur alone or together with cognitive and urinary
symptoms. Adjunct tests, particularly those involving transient removal of
cerebrospinal fluid via lumbar puncture or lumbar drain, can serve the dual
purpose of adding to diagnostic certainty and assisting in prognostication about
response to treatment. Prognostication is important because neurosurgical
treatment by placement of a ventricular shunt, while effective, carries the risk
of potentially significant morbidity. Outcome of shunting in INPH is most often
successful when patients are accurately diagnosed, suitably evaluated for
surgical candidacy, and managed carefully throughout the preoperative, surgical,
and postoperative periods. PURPOSE: We prospectively evaluated the regional cerebral metabolic rate of
glucose (CMRglu) before and after ventricular shunt placement in idiopathic
normal-pressure hydrocephalus (iNPH) patients, to investigate whether some brain
regions are more involved than others; we also correlated the individual
variations of CMRglu with the clinical scale score assessment after shunting.
METHODS: Twenty iNPH patients (12 men; mean age 73 ± 9 years) underwent clinical
scale score assessment and F-FDG PET-CT before and 1 week after shunting.
RESULTS: Before shunting, CMRglu values were similar in right and left brain
regions, as well as after shunting. After shunting, 17 of 20 iNPH patients were
clinically improved; all scale scores decreased, and CMRglu significantly
increased in all regions (P < 10). In 3 of 20 iNPH patients, the symptoms
persisted, the scale scores did not change, and CMRglu increased only in 3
regions: left frontal, left putamen, and right thalamus. Before shunting, no
difference in global CMRglu between clinically improved (n = 17) and not
improved (n = 3) iNPH patients was found. After shunting, a significant (P =
0.01) correlation between individual variations of CMRglu and clinical
assessment was found.
CONCLUSIONS: These findings confirm that iNPH is a disease involving all
cerebral regions almost in the same way, and shunt procedure has a similar
effect on regional cerebral metabolism almost in the same way. Individual
variations of CMRglu are more important than absolute values and correlate with
clinical status after shunting. Clinical improvement depends not only on the
capability to restore the cerebrospinal fluid dynamic, but also on the ability
of cerebral parenchyma to recover the metabolic function. |
Does Serca2a bind PLN in the heart? | Yes, Serca2a bind PLN in the heart. | There is clear evidence for direct regulatory protein-protein interactions
between phospholamban (PLN) and the Ca2+-ATPase of cardiac sarcoplasmic
reticulum (SERCA2a) in cytoplasmic domains, but there is less clear evidence for
regulatory interactions in the transmembrane domains of the two proteins. We
have now coexpressed SERCA isoforms with the transmembrane sequence of PLN and
with epitope-tagged transmembrane sequences of PLN to study intramembrane
interactions in the absence of cytoplasmic interactions. Coexpression of the
transmembrane sequence of phospholamban (Met-PLN28-52) with SERCA1a, SERCA2a,
and SERCA3 inhibited Ca2+ transport by lowering apparent Ca2+ affinity. Addition
of the hemagglutinin (HA) epitope to the transmembrane sequence of PLN
(HA-PLN28-52) or deletion of PLN residues 21-29 (PLN1-20-PLN30-52)
"supershifted" apparent Ca2+ affinity to values lower than those observed with
native PLN without uncoupling Ca2+ transport from ATP hydrolysis. Inhibition by
PLN1-20-PLN30-52 or by Flag-PLN28-52 was reversed by PLN antibody or by Flag
antibody, demonstrating that inhibition by these constructs is reversible and
that the inhibitory constructs are properly oriented in the membrane. These
results suggest that PLN modulates the apparent Ca2+ affinity of SERCA2a through
intramembrane interactions, which are disrupted at long range and in concert
with disruption of the well characterized cytoplasmic interactions. Phospholamban (PLN), a homopentameric, integral membrane protein, reversibly
inhibits cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity through
intramembrane interactions. Here, alanine-scanning mutagenesis of the PLN
transmembrane sequence was used to identify two functional domains on opposite
faces of the transmembrane helix. Mutations in one face diminish inhibitory
interactions with transmembrane sequences of SERCA2a, but have relatively little
effect on the pentameric state, while mutations in the other face activate
inhibitory interactions and enhance monomer formation. Double mutants are
monomeric, but loss of inhibitory function is domit over activation of
inhibitory function. These observations support the proposal that the SERCA2a
interaction site lies on the helical face which is not involved in pentamer
formation. Four highly inhibitory mutants are effectively devoid of pentamer,
suggesting that pentameric PLN represents a less active or inactive reservoir
that dissociates to provide inhibitory monomeric PLN subunits. A model is
presented in which the degree of PLN inhibition of SERCA2a activity is
ultimately determined by the concentration of the inhibited PLN monomer.SERCA2a
heterodimeric complex. The concentration of this inhibited complex is determined
by the dissociation constant for the PLN pentamer (which is mutation-sensitive)
and by the dissociation constant for the PLN/SERCA2a heterodimer (which is
likely to be mutation-sensitive). Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib
in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30
results in gain of inhibitory function. In an earlier study (Kimura, Y.,
Kurzydlowski, K., Tada, M. , and MacLen, D. H. (1997) J. Biol. Chem. 272,
15061-15064), gain of function in PLN transmembrane domain II mutants was
correlated with pentamer destabilization, leading to proposals that the PLN
monomer is the active inhibitory species, that dissociation of the PLN pentamer
is one determit of PLN inhibitory function and that dissociation of the
PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second
determit. Because each of the new domain Ib mutants contained a normal ratio
of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of
function must have resulted from mechanisms other than destabilization of
pentameric structure. Evidence that domain Ib and domain II mutants act through
different sites and different mechanisms was provided by a monomeric double
mutant, N30A/I40A, in which the enhanced inhibitory function of each single
mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a
heterodimer was obtained in a study of double mutant N27A/N34A in which
inhibitory function was regained by combining a gain of function, domain Ib
mutation with a loss of function domain II mutation. These results support the
proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN
and, second, the formation of inhibitory interactions between monomeric PLN and
SERCA2a. Phospholamban (PLN) reversibly inhibits the Ca(2+)-ATPase of cardiac
sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction,
playing a pivotal role in the regulation of intracellular Ca(2+) in heart muscle
cells. The interaction between PLN and SERCA2a occurs at multiple sites within
the cytoplasmic and membrane domains. Here, we have reconstituted the
cytoplasmic protein-protein interaction using bacterially expressed fusion
proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of
SERCA2a. We have developed two methods to evaluate the binding of the fusion
proteins, one with glutathione-Sepharose beads and the other with a 96-well
plate. Essentially the same results were obtained by the two methods. The
affinity of the binding (K(D)) was 0.70 microM. The association was inhibited by
cAMP-dependent phosphorylation of the PLN fusion protein and by usage of
anti-PLN monoclonal antibody. It was also diminished by substitution at the
phosphorylation site of PLN of Ser(16) to Asp. These results suggest that PLN
can bind SERCA2a in the absence of the membrane domains and that the
modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel
the disruption of the association between the two fusion proteins. It has been
shown that the removal of PLN inhibition of SERCA2a rescues cardiac function and
morphology in the mouse dilated cardiomyopathy model. Our assay system can be
applied to the screening of novel inotropic agents that remove the inhibition of
SERCA2a by PLN, improving the relaxation as well as the contractility of the
failing heart. We previously reported that acylphosphatase, a cytosolic enzyme present in
skeletal and heart muscle, actively hydrolyzes the phosphoenzyme (EP) of cardiac
sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), inducing an increased
activity of this pump. We hypothesized that acylphosphatase-induced stimulation
of SERCA2a, in addition to enhanced EP hydrolysis, may be due to a displacement
of phospholamban (PLN), removing its inhibitory effect. To verify this
hypothesis co-immunoprecipitation experiments were performed by adding
recombit muscle acylphosphatase to solubilized heart SR vesicles, used as a
source of SERCA2a and PLN. With anti-acylphosphatase antibodies only SERCA2a was
co-immunoprecipitated in an amount which increased in parallel to the
concentrations of our enzyme. Conversely, using anti-SERCA2a antibody, both PLN
and acylphosphatase were co-immunoprecipitated with SERCA2a, and the PLN amount
in the precipitate decreased with increasing acylphosphatase concentrations.
SERCA2a and PLN were co-immunoprecipitated by anti-phospholamban antibodies, but
while the amount of precipitated phospholamban increased in the presence of
acylphosphatase, the level of SERCA2a decreased. These preliminary results
strengthen the supposed displacement of phospholamban by acylphosphatase. Molecular etiologies of heart failure, an emerging cardiovascular epidemic
affecting 4.7 million Americans and costing 17.8 billion health-care dollars
annually, remain poorly understood. Here we report that an inherited human
dilated cardiomyopathy with refractory congestive heart failure is caused by a
domit Arg --> Cys missense mutation at residue 9 (R9C) in phospholamban
(PLN), a transmembrane phosphoprotein that inhibits the cardiac sarcoplasmic
reticular Ca2+-adenosine triphosphatase (SERCA2a) pump. Transgenic PLN(R9C) mice
recapitulated human heart failure with premature death. Cellular and biochemical
studies revealed that, unlike wild-type PLN, PLN(R9C) did not directly inhibit
SERCA2a. Rather, PLN(R9C) trapped protein kinase A (PKA), which blocked
PKA-mediated phosphorylation of wild-type PLN and in turn delayed decay of
calcium transients in myocytes. These results indicate that myocellular calcium
dysregulation can initiate human heart failure-a finding that may lead to
therapeutic opportunities. Both sarcolipin (SLN) and phospholamban (PLN) lower the apparent affinity of
either SERCA1a or SERCA2a for Ca(2+). Since SLN and PLN are coexpressed in the
heart, interactions among these three proteins were investigated. When SERCA1a
or SERCA2a were coexpressed in HEK-293 cells with both SLN and PLN,
superinhibition resulted. The ability of SLN to elevate the content of PLN
monomers accounts, at least in part, for the superinhibitory effects of SLN in
the presence of PLN. To evaluate the role of SLN in skeletal muscle, SLN cDNA
was injected directly into rat soleus muscle and force characteristics were
analyzed. Overexpression of SLN resulted in significant reductions in both
twitch and tetanic peak force amplitude and maximal rates of contraction and
relaxation and increased fatigability with repeated electrical stimulation.
Ca(2+) uptake in muscle homogenates was impaired, suggesting that overexpression
of SLN may reduce the sarcoplasmic reticulum Ca(2+) store. SLN and PLN appear to
bind to the same regulatory site in SERCA. However, in a ternary complex, PLN
occupies the regulatory site and SLN binds to the exposed side of PLN and to
SERCA. Phospholamban (PLN) is a key regulator of Ca(2+) homeostasis and contractility
in the heart. Its regulatory effects are mediated through its interaction with
the sarcoplasmic reticulum Ca(2+)-ATPase, (SERCA2a), resulting in alterations of
its Ca(2+)-affinity. To identify additional proteins that may interact with PLN,
we used the yeast-two-hybrid system to screen an adult human cardiac cDNA
library. HS-1 associated protein X-1 (HAX-1) was identified as a PLN-binding
partner. The minimal binding regions were mapped to amino acid residues 203-245
for HAX-1 and residues 16-22 for PLN. The interaction between the two proteins
was confirmed using GST-HAX-1, bound to the glutathione-matrix, which
specifically adsorbed native PLN from human or mouse cardiac homogenates, while
in reciprocal binding studies, recombit His-HAX-1 bound GST-PLN. Kinetic
studies using surface plasmon resoce yielded a K(D) of approximately 1 muM as
the binding affinity for the PLN/HAX-1 complex. Phosphorylation of PLN by
cAMP-dependent protein kinase reduced binding to HAX-1, while increasing
concentrations of Ca(2+) diminished the PLN/HAX-1 interaction in a
dose-dependent manner. HAX-1 concentrated to mitochondria, but upon transient
co-transfection of HEK 293 cells with PLN, HAX-1 redistributed and co-localized
with PLN at the endoplasmic reticulum. Analysis of the anti-apoptotic function
of HAX-1 revealed that the presence of PLN enhanced the HAX-1 protective effects
from hypoxia/reoxygenation-induced cell death. These findings suggest a possible
link between the Ca(2+) handling by the sarcoplasmic reticulum and cell survival
mediated by the PLN/HAX-1 interaction. In animal models of conotruncal heart defects, an abnormal calcium sensitivity
of the contractile apparatus and a depressed L-type calcium current have been
described. Sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is a membrane
protein that catalyzes the ATP-dependent transport of Ca(2+) from the cytosol to
the SR. The activity of SERCA is inhibited by phospholamban (PLN) and sarcolipin
(SLN), and all these proteins participate in maintaining the normal
intracellular calcium handling. Ryanodine receptors (RyRs) are the major SR
calcium-release channels required for excitation-contraction coupling in
skeletal and cardiac muscle. Our objective was to evaluate SERCA2a (i.e., the
SERCA cardiac isoform), PLN, SLN, and RyR2 (i.e., the RyR isoform enriched in
the heart) gene expression in myocardial tissue of patients affected by
tetralogy of Fallot (TOF), a conotruncal heart defect. The gene expression of
target genes was assessed semiquantitatively by RT-PCR using the calsequestrin
(CASQ, a housekeeping gene) RNA as internal standard in the atrial myocardium of
23 pediatric patients undergoing surgical correction of TOF, in 10 age-matched
patients with ventricular septal defect (VSD) and in 13 age-matched children
with atrial septal defect (ASD). We observed a significantly lower expression of
PLN and SLN in TOF patients, while there was no difference between the
expression of SERCA2a and RyR2 in TOF and VSD. These data suggest a complex
mechanism aimed to enhance the intracellular Ca(2+) reserve in children affected
by tetralogy of Fallot. Cardiac-type sarco(endo)plasmic reticulum Ca(2)-ATPase (SERCA2a) plays a major
role in cardiac muscle contractility. Phospholamban (PLN) regulates the function
of SERCA2a via its Ser(16)-phosphorylation. Since it has been proposed that the
Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked
N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN
function in rat adult cardiomyocytes. Studies using enzymatic labeling and
co-immunoprecipitation of wild type and a series of mutants of PLN showed that
PLN was O-GlcNAcylated and Ser(16) of PLN might be the site for O-GlcNAcylation.
In cardiomyocytes treated with
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc),
the O-GlcNAcylation was significantly increased compared to non-treated cells.
Simultaneously, Ser(16)-phosphorylation of PLN was reduced. In Chinese hamster
ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected,
the Ser(16)-phosphorylation of PLN was significantly increased compared to
controls. The same results were observed in heart homogenates from diabetic
rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction
between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike
non-treated cells, the activity of SERCA2a and the profiles of calcium
transients in PUGNAc-treated cardiomyocytes were not significantly changed even
after treatment with catecholamine. These data suggest that PLN is
O-GlcNAcylated to induce the inhibition of its phosphorylation, which correlates
to the deterioration of cardiac function. This might define a novel mechanism by
which PLN regulation of SERCA2a is altered under conditions where
O-GlcNAcylation is increased, such as those occurring in diabetes. Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired
sarcoplasmic reticulum (SR) function in human and experimental heart failure.
Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN
mutations in humans have been associated with dilated cardiomyopathy (DCM). We
previously reported the deletion of the highly conserved amino acid residue
arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid
is important in maintaining the upstream consensus sequence for PKA
phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we
introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic
lines expressing mutant PLN-R14Del at similar protein levels to wild types
exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake
compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics
also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO,
and isoproterenol did not further stimulate these hyper-contractile basal
parameters. Consistent with the lack of inhibition on SR Ca-transport and
contractility, confocal microscopy indicated that the PLN-R14Del failed to
co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with
SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal
Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells
indicated significant fluorescence resoce energy transfer between
PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman.
Despite the enhanced cardiac function in PLN-R14Del hearts (as in
PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with
activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to
the sarcolemma, in the absence of endogenous PLN, and alters NKA activity,
leading to cardiac remodeling. The aim of the present study was to make use of the artificially induced aging
model cardiomyocytes to further investigate potential anti-aging-associated
cellular diastolic dysfunction effects of EGB761 and explore underlying
molecular mechanisms. Cultured rat primary cardiomyocytes were treated with
either D-galactose or D-galactose combined with EGB761 for 48 h. After
treatment, the percentage of cells positive for SA-β-gal, AGEs production,
cardiac sarcoplasmic reticulum calcium pump (SERCA) activity, the myocardial
sarcoplasmic reticulum calcium uptake, and relative protein levels were
measured. Our results demonstrated that in vitro stimulation with D-galactose
induced AGEs production. The addition of EGB761 significantly decreased the
number of cells positive for SA-β-gal. Furthermore, decreased diastolic
[Ca(2+)](i), curtailment of the time from the maximum concentration of Ca(2+) to
the baseline level and increased reuptake of Ca(2+) stores in the SR were also
observed. In addition, the level of p-Ser16-PLN protein as well as SERCA was
markedly increased. The study indicated that EGb761 alleviates formation of AGEs
products on SERCA2a in order to mitigate myocardial stiffness on one hand; on
other hand, improve SERCA2a function through increase the amount of Ser16 sites
PLN phosphorylation, which two hands finally led to ameliorate diastolic
dysfunction of aging cardiomyocytes. |
What is the function of the spliceosome complex? | The excision of introns from nascent eukaryotic transcripts is catalyzed by the spliceosome, a highly complex and dynamic macromolecular machine composed of RNA and protein. | Splicing is a crucial, ubiquitous and highly complex step in eukaryotic gene
expression. The daunting complexity of the splicing reaction, although
fascinating, has severely limited our understanding of its mechanistic details.
Recent advances have begun to provide exciting new insights into the dynamic
interactions that govern the function of the spliceosome, the multi-megadalton
complex that performs splicing. An emerging paradigm is the presence of a
succession of distinct conformational states, which are stabilized by an
intricate network of interactions. Recent data suggest that even subtle changes
in the composition of the interaction network can result in interconversion of
the different conformational states, providing opportunities for regulation and
proofreading of spliceosome function. Significant progress in proteomics has
elucidated the protein composition of the spliceosome at different stages of
assembly. Also, the increased sophistication and resolution of cryo-electron
microscopy techniques, combined with high-resolution structural studies on a
smaller scale, promise to create detailed images of the global structure of the
spliceosome and its main components, which in turn will provide a plethora of
mechanistic insights. Overall, the past two years have seen a convergence of
data from different lines of research into what promises to become a holistic
picture of spliceosome function. The excision of introns from nascent eukaryotic transcripts is catalyzed by the
spliceosome, a highly complex and dynamic macromolecular machine composed of RNA
and protein. Because of its complexity, biochemical analysis of the spliceosome
has been previously limited to bulk assays in largely unfractionated cell
extracts. We now report development of methodologies for studying the splicing
of isolated single pre-mRNA molecules in real time. In this system, a
fluorescently tagged pre-mRNA is tethered to a glass surface via its 3'-end.
Splicing can be observed in Saccharomyces cerevisiae whole cell extract by
monitoring loss of intron-specific fluorescence with a multi-wavelength total
internal reflection fluorescence (TIRF) microscope. To prolong fluorophore
lifetime, two enzyme-based O2 scavenging systems compatible with splicing were
also developed. This work provides a powerful new approach for elucidating the
mechanisms of spliceosome function and demonstrates the feasibility of utilizing
TIRF microscopy for biochemical studies of single molecules in highly complex
environments. Spliceosomes are macro-complexes involving hundreds of proteins with many
functional interactions. Spliceosome assembly belongs to the key processes that
enable splicing of mRNA and modulate alternative splicing. A detailed list of
factors involved in spliceosomal reactions has been assorted over the past
decade, but, their functional interplay is often unknown and most of the present
biological models cover only parts of the complete assembly process. It is a
challenging task to build a computational model that integrates dispersed
knowledge and combines a multitude of reaction schemes proposed earlier.Because
for most reactions involved in spliceosome assembly kinetic parameters are not
available, we propose a discrete modeling using Petri nets, through which we are
enabled to get insights into the system's behavior via computation of structural
and dynamic properties. In this paper, we compile and examine reactions from
experimental reports that contribute to a functional spliceosome. All these
reactions form a network, which describes the inventory and conditions necessary
to perform the splicing process. The analysis is mainly based on system
invariants. Transition invariants (T-invariants) can be interpreted as signaling
routes through the network. Due to the huge number of T-invariants that arise
with increasing network size and complexity, maximal common transition sets
(MCTS) and T-clusters were used for further analysis. Additionally, we introduce
a false color map representation, which allows a quick survey of network modules
and the visual detection of single reactions or reaction sequences, which
participate in more than one signaling route. We designed a structured model of
spliceosome assembly, which combines the demands on a platform that i) can
display involved factors and concurrent processes, ii) offers the possibility to
run computational methods for knowledge extraction, and iii) is successively
extendable as new insights into spliceosome function are reported by
experimental reports. The network consists of 161 transitions (reactions) and
140 places (reactants). All reactions are part of at least one of the 71
T-invariants. These T-invariants define pathways, which are in good agreement
with the current knowledge and known hypotheses on reaction sequences during
spliceosome assembly, hence contributing to a functional spliceosome. We
demonstrate that present knowledge, in particular of the initial part of the
assembly process, describes parallelism and interaction of signaling routes,
which indicate functional redundancy and reflect the dependency of spliceosome
assembly initiation on different cellular conditions. The complexity of the
network is further increased by two switches, which introduce alternative routes
during A-complex formation in early spliceosome assembly and upon transition
from the B-complex to the C-complex. By compiling known reactions into a
complete network, the combinatorial nature of invariant computation leads to
pathways that have previously not been described as connected routes, although
their constituents were known. T-clusters divide the network into modules, which
we interpret as building blocks in spliceosome maturation. We conclude that
Petri net representations of large biological networks and system invariants,
are well-suited as a means for validating the integration of experimental
knowledge into a consistent model. Based on this network model, the design of
further experiments is facilitated. BACKGROUND: Splicing and alternate splicing are the two key biological processes
that result in the generation of diverse transcript and protein isoforms in
Plasmodium falciparum as well as in other eukaryotic organisms. Not much is
known about the organization of splicing machinery and mechanisms in human
malaria parasite. Present study reports the organization and assembly of
Plasmodium spliceosome Sm core complex.
METHODS: Presence of all the seven Plasmodium Sm-like proteins in the
intra-erythrocytic stages was assessed based on the protein(s) expression
analysis using immuno-localization and western blotting.
Localization/co-localization studies were performed by immunofluorescence
analysis on thin parasite smear using laser scanning confocal microscope.
Interaction studies were carried out using yeast two-hybrid analysis and
validated by in vitro pull-down assays. PfPRMT5 (arginine methyl transferase)
and PfSmD1 interaction analysis was performed by pull-down assays and the
interacting proteins were identified by MALDI-TOF spectrometry.
RESULTS: PfSm proteins are expressed at asexual blood stages of the parasite and
show nucleo-cytoplasmic localization. Protein-protein interaction studies showed
that PfSm proteins form a heptameric complex, typical of spliceosome core
complex as shown in humans. Interaction of PfSMN (survival of motor neuron,
tudor domain containing protein) or PfTu-TSN (Tudor domain of Tudor
Staphylococcal nuclease) with PfSmD1 proteins was found to be methylation
dependent. Co-localization by immunofluorescence and co-immunoprecipitation
studies suggested an association between PfPRMT5 and PfSmD1, indicating the role
of arginine methylation in assembly of Plasmodium spliceosome complex.
CONCLUSIONS: Plasmodium Sm-like proteins form a heptameric ring-like structure,
although the arrangement of PfSm proteins slightly differs from human splicing
machinery. The data shows the interaction of PfSMN with PfSmD1 and this
interaction is found to be methylation dependent. PfPRMT5 probably exists as a
part of methylosome complex that may function in the cytoplasmic assembly of Sm
proteins at asexual blood stages of P. falciparum. The spliceosome machinery is composed of multimeric protein complexes that
generate a diverse repertoire of mRNA through coordinated splicing of
heteronuclear RNAs. While somatic mutations in spliceosome components have been
discovered in several cancer types, the molecular bases and consequences of
spliceosome aberrations in cancer are poorly understood. Here we report for the
first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein)
spliceosome complex, drives cancer proliferation by preferential splicing of
genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP
complex proteins, but not other snRNP spliceosome complexes, selectively
abrogated growth in cancer cells with high tri-snRNP levels. High-resolution
transcriptome analyses revealed that reduced PRPF6 alters the constitutive and
alternative splicing of a discrete number of genes, including an oncogenic
isoform of the ZAK kinase. These findings implicate an essential role for PRPF6
in cancer via splicing of distinct growth-related gene products. |
Is there any role for long noncoding RNAs in adipogenesis? | Yes. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, numerous lncRNAs are functionally required for proper adipogenesis. | The prevalence of obesity has led to a surge of interest in understanding the
detailed mechanisms underlying adipocyte development. Many protein-coding genes,
mRNAs, and microRNAs have been implicated in adipocyte development, but the
global expression patterns and functional contributions of long noncoding RNA
(lncRNA) during adipogenesis have not been explored. Here we profiled the
transcriptome of primary brown and white adipocytes, preadipocytes, and cultured
adipocytes and identified 175 lncRNAs that are specifically regulated during
adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during
adipogenesis, and bound at their promoters by key transcription factors such as
peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding
protein α (CEBPα). RNAi-mediated loss of function screens identified functional
lncRNAs with varying impact on adipogenesis. Collectively, we have identified
numerous lncRNAs that are functionally required for proper adipogenesis. |
Which genes code for the alpha subunit of the DNA polymerase III in most Firmicutes? | Bacterial DNA polymerase III is the primary complex of DNA replication. In most Firmicutes, which are low-GC, gram-positive bacteria, the alpha subunit of their DNA polymerase III is encoded by polC and dnaE. DnaE is widely conserved in most bacteria, while PolC is present mainly in Firmicutes clade. | The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the
DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs
coding for 1335 amino acids (from the start to the stop codon), giving a
molecular weight of 151,273. A mutation (azp-12) that confers resistance to the
antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change
at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino
acid, serine, to alanine. It is in the active site and located at the C-terminal
part of the enzyme. The amino acid composition in an N-terminal domain has 26%
homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli,
which is a 3'----5' proofreading exonuclease, supporting an earlier observation
that this function is an integral part of the polymerase molecule in B.
subtilis. pSM19035 is a low-copy-number theta-replicating plasmid, which belongs to the
Inc18 family. Plasmids of this family, which show a modular organization, are
functional in evolutionarily diverse bacterial species of the Firmicutes Phylum.
This review summarizes our understanding, accumulated during the last 20 years,
on the genetics, biochemistry, cytology and physiology of the five pSM19035
segregation (seg) loci, which map outside of the minimal replicon. The segA
locus plays a role both in maximizing plasmid random segregation, and in
avoiding replication fork collapses in those plasmids with long inverted
repeated regions. The segB1 locus, which acts as the ultimate determit of
plasmid maintece, encodes a short-lived epsilon(2) antitoxin protein and a
long-lived zeta toxin protein, which form a complex that neutralizes zeta
toxicity. The cells that do not receive a copy of the plasmid halt their
proliferation upon decay of the epsilon(2) antitoxin. The segB2 locus, which
encodes two trans-acting, ParA- and ParB-like proteins and six cis-acting parS
centromeres, actively ensures equal or roughly equal distribution of plasmid
copies to daughter cells. The segC locus includes functions that promote the
shift from the use of DNA polymerase I to the replicase (PolC-PolE DNA
polymerases). The segD locus, which encodes a trans-acting transcriptional
repressor, omega(2), and six cis-acting cognate sites, coordinates the
expression of genes that control copy number, better-than-random segregation and
partition, and assures the proper balance of these different functions. Working
in concert the five different loci achieve almost absolute plasmid maintece
with a minimal growth penalty. BACKGROUND: Bacterial genomes displaying a strong bias between the leading and
the lagging strand of DNA replication encode two DNA polymerases III, DnaE and
PolC, rather than a single one. Replication is a highly unsymmetrical process,
and the presence of two polymerases is therefore not unexpected. Using
comparative genomics, we explored whether other processes have evolved in
parallel with each polymerase.
RESULTS: Extending previous in silico heuristics for the analysis of gene
co-evolution, we analyzed the function of genes clustering with dnaE and polC.
Clusters were highly informative. DnaE co-evolves with the ribosome, the
transcription machinery, the core of intermediary metabolism enzymes. It is also
connected to the energy-saving enzyme necessary for RNA degradation,
polynucleotide phosphorylase. Most of the proteins of this co-evolving set
belong to the persistent set in bacterial proteomes, that is fairly ubiquitously
distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are
present only in the A+T-rich Firmicutes clade, suggesting at least two origins
for the degradosome.
CONCLUSION: DNA replication involves two machineries, DnaE and PolC. DnaE
co-evolves with the core functions of bacterial life. In contrast PolC
co-evolves with a set of RNA degradation enzymes that does not derive from the
degradosome identified in gamma-Proteobacteria. This suggests that at least two
independent RNA degradation pathways existed in the progenote community at the
end of the RNA genome world. The mechanism of DNA replication is one of the driving forces of genome
evolution. Bacterial DNA polymerase III, the primary complex of DNA replication,
consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria,
especially in the Firmicutes with low GC content, whereas DnaE is widely
conserved in most Gram-negative and Gram-positive bacteria. PolC contains two
domains, the 3'-5'exonuclease domain and the polymerase domain, while DnaE only
possesses the polymerase domain. Accordingly, DnaE does not have the
proofreading function; in Escherichia coli, another enzyme DnaQ performs this
function. In most bacteria, the fidelity of DNA replication is maintained by
3'-5' exonuclease and a mismatch repair (MMR) system. However, we found that
most Actinobacteria (a group of Gram-positive bacteria with high GC content)
appear to have lost the MMR system and chromosomes may be replicated by
DnaE-type DNA polymerase III with DnaQ-like 3'-5' exonuclease. We tested the
mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found
that the wild type strain is AT-biased while the mutS-deletant strain is
remarkably GC-biased. If we presume that DnaE tends to make mistakes that
increase GC content, these results can be explained by the mutS deletion (i.e.,
deletion of the MMR system). Thus, we propose that GC content is regulated by
DNA polymerase and MMR system, and the absence of polC genes, which participate
in the MMR system, may be the reason for the increase of GC content in
Gram-positive bacteria such as Actinobacteria. |
Is there any research that relates the function of Notch Signaling with Alzheimer Disease? | Notch signaling is an evolutionarily conserved pathway, which is fundamental for neuronal development and specification. In the last decade, increasing evidence has pointed out an important role of this pathway beyond embryonic development, indicating that Notch also displays a critical function in the mature brain of vertebrates and invertebrates. This pathway appears to be involved in neural progenitor regulation, neuronal connectivity, synaptic plasticity and learning/memory. In addition, Notch appears to be aberrantly regulated in neurodegenerative diseases, including Alzheimer's disease and ischemic injury | Understanding complex diseases such as sporadic Alzheimer disease (AD) has been
a major challenge. Unlike the familial forms of AD, the genetic and
environmental risks factors identified for sporadic AD are extensive. MicroRNAs
are one of the major noncoding RNAs that function as negative regulators to
silence or suppress gene expression via translational inhibition or message
degradation. Their discovery has evoked great excitement in biomedical research
for their promise as potential disease biomarkers and therapeutic targets. Key
microRNAs have been identified as essential for a variety of cellular events
including cell lineage determination, proliferation, apoptosis, DNA repair, and
cytoskeletal organization; most, if not all, acting to fine-tune gene expression
at the post-transcriptional level in a host of cellular signaling networks.
Dysfunctional microRNA-mediated regulation has been implicated in the
pathogenesis of many disease states. Here, the current understanding of the role
of miRNAs in the central nervous system is reviewed with emphasis on their
impact on the etiopathogenesis of sporadic AD. The γ-secretase complex is responsible for intramembrane processing of over 60
substrates and is involved in Notch signaling as well as in the generation of
the amyloid β-peptide (Aβ). Aggregated forms of Aβ have a pathogenic role in
Alzheimer disease and, thus, reducing the Aβ levels by inhibiting γ-secretase is
a possible treatment strategy for Alzheimer disease. Regrettably, clinical
trials have shown that inhibition of γ-secretase results in Notch-related side
effects. Therefore, it is of great importance to find ways to inhibit amyloid
precursor protein (APP) processing without disturbing vital signaling pathways
such as Notch. Nicastrin (Nct) is part of the γ-secretase complex and has been
proposed to be involved in substrate recognition and selection. We have
investigated how the four evenly spaced and conserved cysteine residues in the
Nct ectodomain affect APP and Notch processing. We mutated these cysteines to
serines and analyzed them in cells lacking endogenous Nct. We found that two
mutants, C213S (C2) and C230S (C3), differentially affected APP and Notch
processing. Both the formation of Aβ and the intracellular domain of amyloid
precursor protein (AICD) were reduced, whereas the production of Notch
intracellular domain (NICD) was maintained on a high level, although C230S (C3)
showed impaired complex assembly. Our data demonstrate that single residues in a
γ-secretase component besides presenilin are able to differentially affect APP
and Notch processing. |
Describe the known functions for the prothymosin alpha c-terminal peptide? | Prothymosin alpha (ProTα) (encoded in human by the PTMA gene) is a ubiquitous, highly acidic nuclear polypeptide. During early apoptosis, proTα is cleaved by activated caspase-3, with a primary attach site being D99, close to its carboxyl-terminus. The role of the cleaved decapeptide -- proTα(100-109) -- is not fully understood. proTα(100-109), which contains the nuclear localization signal (NLS) for ProTα, has been demonstrated to have immunostimulatory properties, such as to stimulate lymphocytes and neutrophils and induce dendritic cell maturation. | A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and
characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha
(fProt-alpha) predicted a 109 amino acid protein with a high homology to the
mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid
residues and presents the typical basic KKQK amino acid sequence in the close
carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is
highly expressed in the testis. A different expression of fProt-alpha transcript
was found during the frog reproductive cycle with a peak in September/October in
concomitance with germ cell maturation, strongly suggesting a role for this
protein in the testicular activity. In situ hybridization evidenced that the
only germ cells expressing fProt-alpha are the primary and secondary
spermatocytes; in addition, the hybridization signal was stronger in the October
testis. Taken together, our findings indicate that fProt-alpha might contribute
to the efficiency of frog spermatogenesis with a role during the meiosis. This
study is the first report on the isolation and characterization of a Prot-alpha
in a non-mammalian vertebrate. In addition, our results indicate that the testis
of the frog R. esculenta may be a useful model to increase the knowledge
concerning the physiological role of Prot-alpha in vertebrates. Prothymosin alpha (proTalpha) is a 109 amino acid long polypeptide presenting
distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest
that in apoptotic cells, proTalpha is cleaved by caspases at its
carboxy(C)-terminus generating potentially bioactive fragments. In this study,
we identified the peptide segment of proTalpha presenting maximum
immunomodulatory activity. Calf thymus proTalpha was trypsinised, and the five
fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109)
were tested for their ability to stimulate healthy donor- and cancer
patient-derived peripheral blood mononuclear cell (PBMC) proliferation in
autologous mixed lymphocyte reaction (AMLR), natural killer and
lymphokine-activated killer cell activity, intracellular production of perforin,
upregulation of adhesion molecules and CD25 expression. ProTalpha(89-102) and
proTalpha(103-109) significantly fortified healthy donor-lymphocytes' immune
responses to levels comparable to those induced by intact proTalpha. These
effects were more pronounced in cancer patients, where peptides
proTalpha(89-102) and proTalpha(103-109) partly, however significantly, restored
the depressed AMLR and cytolytic ability of PBMC, by simulating the biological
activity exerted by intact proTalpha. ProTalpha(1-14), proTalpha(21-30) and
proTalpha(31-87) marginally upregulated lymphocyte activation. This is the first
report showing that proTalpha's immunomodulating activity can be substituted by
its C-terminal peptide(s). Whether generation and externalization of such
immunoactive proTalpha fragments occurs in vivo, needs further investigation.
However, if these peptides can trigger immune responses, they may eventually be
used therapeutically to improve some PBMC functions of cancer patients. Human thymosin alpha 1 (Talpha1) is an important peptide in the development and
senescence of immunological competence in human, and many studies have reported
the expression of this peptide. In this study, we designed and synthesized the
Talpha1 gene according to the E. coli codon usage preference and constructed a
6xTalpha1 concatemer. The latter was inserted into an E. coli expression vector
pET-22b (+), and transformed into E. coli BL21 (DE3). After induction with IPTG,
the concatemer protein was successfully expressed in E. coli then cleaved by
hydroxylamine to release the Talpha1 monomer. Gly-SDS-PAGE and mass spectrometry
confirmed that the recombit protein was cleaved as intended. The bioactivity
of the Talpha1 monomer was analyzed by lymphocyte proliferation and by
mitochondrial activity in two different tumor cell lines. This study provides a
description of the preparation of a bioactive Talpha1, which may prove useful in
future biomedical research. The nuclear protein prothymosin-α (ProTα), which lacks a signal peptide
sequence, is released from neurons and astrocytes on ischemic stress and exerts
a unique form of neuroprotection through an anti-necrotic mechanism. Ischemic
stress-induced ProTα release is initiated by a nuclear release, followed by
extracellular release in a non-vesicular manner, in C6 glioma cells. These
processes are caused by ATP loss and elevated Ca²(+), respectively. S100A13, a
Ca²(+)-binding protein, was identified to be a major protein co-released with
ProTα in an immunoprecipitation assay. The Ca²(+)-dependent interaction between
ProTα and S100A13 was found to require the C-terminal peptide sequences of both
proteins. In C6 glioma cells expressing a Δ88-98 mutant of S100A13, serum
deprivation caused the release of S100A13 mutant, but not of ProTα. When cells
were administered apoptogenic compounds, ProTα was cleaved by caspase-3 to
generate a C-terminal peptide-deficient fragment, which lacks the nuclear
localization signal (NLS). However, there was no extracellular release of ProTα.
All these results suggest that necrosis-inducing stress induces an extacellular
release of ProTα in a non-vesicular manner, whereas apoptosis-inducing stress
does not, owing to the loss of its interaction with S100A13, a cargo molecule
for extracellular release. Neutrophils are short-lived leukocytes and major components of the innate immune
system. They are key players in the body's defense against pathogens, but their
contribution to tumor growth and metastasis is controversial. Nevertheless,
improving the functions of neutrophils in cancer patients, particularly in those
undergoing chemotherapy, is of clinical significance. In this study, we
investigated the ability of the immunoreactive fragment of the polypeptide
prothymosin alpha (proTα), i.e., the decapeptide proTα(100-109), to enhance the
functions of neutrophils isolated from the peripheral blood of breast cancer
patients in comparison with those from healthy donors. Activation of neutrophils
from both groups with proTα(100-109) significantly increased phagocytosis and
the production of intracellular reactive oxygen species (ROS) compared to
controls. The release of extracellular ROS and oxidative burst of
proTα(100-109)-stimulated neutrophils were less improved. Most importantly, upon
activation with proTα(100-109), neutrophils from breast cancer patients showed
significantly enhanced cytotoxicity against tumor cell targets. Using a
scrambled peptide as a control, we showed that the proTα(100-109)-induced
effects were sequence-specific and comparable to those exerted by the parental
molecule proTα. The responsiveness of neutrophils to proTα(100-109) or intact
proTα did not correlate with the tumor grade or other established tumor
characteristics. Our results suggest that proTα(100-109) activates neutrophils,
particularly those derived from breast cancer patients, and these effects could
potentially be used to improve some functions of neutrophils in the clinical
setting. |
Was modafinil tested for schizophrenia treatment? | Yes. Modafinil has been shown to improve attention, memory, executive function and antipsychotic-induced parkinsonism in patients with schizophrenia. However, some authors have failed to demonstrate beneficial action of modafinil for schizophrenia. | Modafinil, a novel cognitive enhancer, selectively improves neuropsychological
task performance in healthy volunteers and adult patients with attention deficit
hyperactivity disorder (ADHD). It has been argued that persistent cognitive
deficits in patients with schizophrenia are responsible for the failure of many
patients to rehabilitate socially even when psychotic symptoms are in remission.
The present study examined the potential of modafinil as a cognitive enhancer in
schizophrenia. Twenty chronic patients with a diagnosis of schizophrenia were
entered into a double-blind, randomized, placebo-controlled crossover study
using a 200 mg dose of modafinil. Modafinil had some cognitive enhancing
properties in schizophrenia similar to those observed in healthy adults and
adult patients with ADHD. Improvement was seen on short-term verbal memory span,
with trends towards improved visual memory and spatial planning. This was
accompanied by slowed response latency on the spatial planning task. No effect
on stop-signal performance was seen. Importantly, significant improvement in
attentional set shifting was seen, despite no effect of modafinil on this task
being seen in healthy volunteers or ADHD patients. Modafinil may have potential
as an important therapy for cognitive impairment in patients with schizophrenia,
particularly because of its beneficial effects on attentional set shifting. Patients with schizophrenia experience cognitive impairments associated with
hypofunctioning of the frontal cortex. Modafinil, a novel wake-promoting agent,
works through the sleep-wake centers of the brain to activate the cortex. This
4-week, open-label, pilot study evaluated adjunct modafinil in patients with
schizophrenia or schizoaffective disorder. Eleven patients received once-daily
oral doses of modafinil (100 mg/day, days 1-14; 100 or 200 mg/day, days 15-28)
in addition to antipsychotic therapy. Modafinil significantly improved patients'
global functioning as assessed by a blinded clinician (week 2, P = 0.026; week
4, P = 0.012) and the investigator (week 3, P = 0.035). Modafinil significantly
improved overall clinical condition, with 64% and 82% of patients rated as
clinically improved at week 4 by a blinded clinician and the investigator
respectively. Eighty-nine percent of patients considered themselves to be
clinically improved. Modafinil significantly improved fatigue (P = 0.025, week
3) and tended to improve cognitive functioning scores. Control of positive
symptoms was well maintained. Treatment-emergent adverse events included dry
mouth (n = 2) and hallucinations (n = 2). One patient discontinued the study
because of hallucinations that were considered to be possibly related to
inadequate antipsychotic therapy. Although preliminary, these results suggest
modafinil may be an effective and well-tolerated adjunct treatment that improves
global functioning and clinical condition, and reduces fatigue in patients with
schizophrenia or schizoaffective disorder. Additional controlled studies are
warranted. BACKGROUND: Schizophrenia is associated with widespread cognitive deficits that
have an impact on social function. Modafinil promotes wakefulness and is
reported to enhance cognition.
AIMS: To study the acute effects of modafinil administration upon brain activity
and cognitive performance in people with chronic schizophrenia.
METHOD: In a randomised double-blind placebo-controlled crossover design, 19
patients received either modafinil (100 mg) or placebo prior to undertaking a
working memory task with functional magnetic resoce imaging.
RESULTS: Seventeen patients completed the study and another underwent acute
relapse 4 days post-drug. Modafinil administration was associated with
significantly greater activation in the anterior cingulate cortex during the
working memory task. The anterior cingulate cortex signal correlated with
cognitive performance, although only a subset of patients exhibited
'enhancement'.
CONCLUSIONS: Modafinil modulates anterior cingulate cortex function in chronic
schizophrenia but its beneficial cognitive effects may be restricted to a subset
of patients requiring further characterisation. OBJECTIVE: To assess the effects of modafinil on fatigue, symptoms, attention,
working memory, and executive functioning in schizophrenia patients treated with
psychotropic medications.
METHOD: Twenty-four patients with a DSM-IV diagnosis of schizophrenia or
schizoaffective disorder (10 men and 14 women) were randomly assigned to
modafinil up to 200 mg a day (N = 13) or placebo (N = 11) as an adjunct therapy
in an 8-week, double-blind, placebo-controlled study. Data were collected from
May 18, 2001 to September 11, 2003.
RESULTS: Four subjects terminated the study early, including one because of
worsening of psychosis during the first week taking modafinil. In the modafinil
(N = 10) and placebo (N = 10) groups, fatigue improved significantly over time
(p < .01), but there were no differences between groups on changes in fatigue,
positive and negative symptoms, or cognition.
CONCLUSION: Fatigue improved in both groups, and there were no differences
between groups on changes in fatigue, symptoms, attention, working memory, or
executive functioning. Lack of differences between groups may be due to small
sample size or possible regression to the mean in the placebo group. BACKGROUND: There are virtually no controlled data suggesting that concomitant
psychotropic medications (CPMs) improve outcome in schizophrenia after the acute
phase. Despite that, polypharmacy (with all of its disadvantages) is far more
common than monotherapy. To our knowledge, there have been no published reports
of prospective systematic investigations of the efficacy of unrestricted CPM use
in nonacute schizophrenia.
METHOD: This was a naturalistic, systematic study using a sample of 53
stabilized patients with DSM-IV-TR schizophrenia from 1 clinical practice
setting including both private patients and patients from controlled research
studies of the effectiveness of antipsychotics. Since there are meager
controlled or systematic data on the effectiveness of CPM use with
antipsychotics in nonacute schizophrenia, we tested the clinical strategy of CPM
use by gradually tapering all CPMs (except antianxiety agents). The aim was to
determine if the CPM improved outcome, had no effect, or worsened outcome using
the Clinical Global Impressions-Improvement scale before and after taper, over
at least 3 months and in some cases up to 18 months after discontinuation. Data
were gathered from July 2002 to June 2005.
RESULTS: For 21 patients undergoing 22 antidepressant tapers, no change was
noted in 18 of 22 tapers, while in 3 improvement was noted and in 1 worsening
was noted. For the 12 patients on treatment with mood stabilizers, no change was
noted in 10 of 13 discontinuations, while in 3 mild worsening was noted. One
patient was on treatment with both modafinil and trazodone and reported no
change after tapering each in separate discontinuation trials, while another 3
patients were taking sleeping medications and also noted no change after
discontinuation.
CONCLUSION: For most stabilized, chronic patients with schizophrenia, tapering
adjunctive medications did not change outcome. This naturalistic study further
defines the limits of efficacy of some concomitant classes of medications in
patients with chronic schizophrenia who are already receiving adequate
antipsychotic therapy. Avolition affects quality of life in chronic schizophrenia. We investigated the
effect of modafinil upon unconstrained motor activity in 18 male patients. In a
randomised crossover design study, wrist-worn actigraphic monitors were used to
objectively record motor activity over a 20 h period. Patients' total activity
was significantly greater when given the drug. These data suggest that modafinil
increases quantifiable motor behaviour in schizophrenia and may have an impact
on avolition. OBJECTIVE: Negative symptoms are core features of schizophrenia that are
functionally debilitating, associated with poor outcomes, and resistant to
existing pharmacotherapies. We performed a randomized, double-blind,
placebo-controlled study of modafinil, a medication approved for the treatment
of excessive daytime sleepiness, to explore its efficacy as an adjunctive
therapy for negative symptoms in schizophrenia.
METHOD: Twenty subjects with DSM-IV schizophrenia or schizoaffective disorder
were randomly assigned to double-blind treatment with modafinil or placebo for 8
weeks. The study ran from March 2002 through March 2006. Outcome measures
included the Scale for the Assessment of Negative Symptoms (SANS), Brief
Psychiatric Rating Scale (BPRS), Clinical Global Impressions (CGI) scale,
Quality of Life Interview, neurocognitive assessments (California Verbal
Learning Test, Degraded Performance-Continuous Performance Test, Trail-Making
Test B), and somatic measures (sleep, weight, side effects).
RESULTS: Modafinil treatment was associated with a greater rate (CGI-Improvement
[CGI-I] score < or = 3, 7/10 vs. 1/10) and degree (mean CGI-I score, 3.2 vs.
4.1) of global improvement at study endpoint compared with placebo. However,
modafinil did not significantly improve global negative symptoms as measured by
the total SANS or SANS individual global items. Modafinil did not significantly
worsen psycho-pathology (according to the BPRS), compared with placebo, and was
well tolerated.
CONCLUSIONS: Although no effect on negative symptoms was found, adjunctive
therapy with modafinil may result in global improvements in patients with
schizophrenia who have prominent negative symptoms. Modafinil, a wake-promoting agent believed to operate via the hypocretin/orexin
system, has a similar clinical profile to that of conventional, dopaminergic
stimulants but different biochemical and pharmacological properties. There is
increasing interest in the use of modafinil to improve cognition in
schizophrenia as well as in other disorders such as
attention-deficit/hyperactivity disorder. Recent research has focused on
enhancing cognition in patients with schizophrenia because of the association
between cognitive performance and functional outcome. Initial findings indicate
that modafinil may lead to better executive functioning and attentional
performance in patients with schizophrenia. The results further suggest that
patient characteristics such as overall current cognitive functioning levels,
genetic polymorphisms, and medication status may be important mediators for the
effectiveness of modafinil, allowing for future treatment to be targeted to
those most likely to benefit. Currently, further research is required to address
the potential benefits and risks of chronic administration of modafinil to
patients with schizophrenia. Modafinil (2-[(Diphenylmethyl) sulfinyl] acetamide, Provigil) is an FDA-approved
medication with wake-promoting properties. Pre-clinical studies of modafinil
suggest a complex profile of neurochemical and behavioral effects, distinct from
those of amphetamine. In addition, modafinil shows initial promise for a variety
of off-label indications in psychiatry, including treatment-resistant
depression, attention-deficit/hyperactivity disorder, and schizophrenia.
Cognitive dysfunction may be a particularly important emerging treatment target
for modafinil, across these and other neuropsychiatric disorders. We aimed to
comprehensively review the empirical literature on neurochemical actions of
modafinil, and effects on cognition in animal models, healthy adult humans, and
clinical populations. We searched PubMed with the search term 'modafinil' and
reviewed all English-language articles for neurochemical, neurophysiological,
cognitive, or information-processing experimental measures. We additionally
summarized the pharmacokinetic profile of modafinil and clinical efficacy in
psychiatric patients. Modafinil exhibits robust effects on catecholamines,
serotonin, glutamate, gamma amino-butyric acid, orexin, and histamine systems in
the brain. Many of these effects may be secondary to catecholamine effects, with
some selectivity for cortical over subcortical sites of action. In addition,
modafinil (at well-tolerated doses) improves function in several cognitive
domains, including working memory and episodic memory, and other processes
dependent on prefrontal cortex and cognitive control. These effects are observed
in rodents, healthy adults, and across several psychiatric disorders.
Furthermore, modafinil appears to be well-tolerated, with a low rate of adverse
events and a low liability to abuse. Modafinil has a number of neurochemical
actions in the brain, which may be related to primary effects on
catecholaminergic systems. These effects are in general advantageous for
cognitive processes. Overall, modafinil is an excellent candidate agent for
remediation of cognitive dysfunction in neuropsychiatric disorders. RATIONALE: Selective cognitive impairments, including those of executive
function as assessed using the Wisconsin Card Sort Test or
intradimensional-extradimensional (ID-ED) tests, are a key feature of
schizophrenia but remain inadequately treated by existing therapies. Recently,
however, modafinil has been shown to improve attentional set-shifting
performance in patients with schizophrenia.
OBJECTIVE: The present study evaluated the recently described analogous rat
ID-ED attentional set-shifting task by investigating the effects of various
pharmacological challenges to a phencyclidine (PCP)-induced ED shift impairment,
namely, haloperidol, risperidone, sertindole, and modafinil.
MATERIALS AND METHODS: Rats were subjected to a subchronic systemic
administration of either saline vehicle or PCP (5 mg/kg i.p. b.i.d. for 7 days)
followed by a 7-day washout period. During this period, rats were trained to dig
in baited bowls for a food reward and to discriminate based on odor or digging
media. In a single test session conducted the day after the washout period (day
8), rats performed a series of discriminations following acute administration of
either vehicle, or haloperidol (0.1 mg/kg s.c.), or risperidone (0.2 mg/kg
i.p.), or sertindole (1.25 mg/kg p.o.) or modafinil (64 mg/kg p.o.).
RESULTS: The subchronic PCP-induced ED deficit was ameliorated by sertindole and
modafinil but not by haloperidol or risperidone.
CONCLUSIONS: Overall, these findings further support that the rat ID-ED test in
subchronic PCP-treated rats has utility and validity as a preclinical model of
the cognitive symptoms of schizophrenia and demonstrates back-translational
potential. RATIONALE: The wake-promoting agent modafinil selectively improves
neuropsychological task performance in healthy volunteers, in adults with
attention deficit hyperactivity disorder (ADHD) and in schizophrenia. We
examined whether modafinil induced similar effects in individuals with
Huntington's disease (HD).
MATERIALS AND METHODS: Twenty patients with genetically proven, mild HD
participated in a double-blind, randomised, placebo-controlled cross-over study
using a single 200 mg dose of modafinil. Patients undertook a battery of
neuropsychological tests including measures of cognition and mood.
RESULTS: Modafinil increased alertness as indexed by visual analogue scales.
Modafinil did not elicit any significant improvements in cognitive function or
mood. Modafinil had a deleterious effect on visual recognition and working
memory.
CONCLUSIONS: Two hundred milligrams acute modafinil administration did improve
alertness but did not improve cognition or mood in patients with mild HD. A
multiple dose, chronic administration study is needed before the potential
clinical utility of modafinil in HD is discounted. OBJECTIVE: Given recent reports about the off-label use of modafinil as an
adjuvant for the treatment of antipsychotic-associated sedation in schizophrenia
patients and the recent interest in its putative cognitive-enhancing effects in
this population, we present a systematic review of available data on trials of
modafinil as an adjuvant in the treatment of cognitive deficits, negative
symptoms, and antipsychotic-induced fatigue, and its tolerability.
DATA SOURCES: PubMed was searched for trials published in English up to January
2008 evaluating modafinil's effects on fatigue, negative symptoms, and cognition
in schizophrenia with combinations of the following terms: schizophrenia,
modafinil, cognition, negative symptoms, and fatigue.
STUDY SELECTION: Six trials were identified: 2 randomized, prospective,
double-blind placebo-controlled trials; 3 randomized, prospective, double-blind
placebo-controlled crossover trials; and 1 open-label pilot study. Case series
and case reports were excluded in the data analysis, except to identify
potential adverse reactions to modafinil.
DATA EXTRACTION: Studies were examined for number of subjects, trial duration,
design, dosing, and outcomes with respect to sedation, negative symptoms,
cognitive function, and tolerability.
RESULTS: One of 4 reviewed studies found a significant effect of modafinil as an
alerting agent for antipsychotic-induced fatigue and sedation. Neither of 2
reviewed studies found modafinil to improve negative symptoms of schizophrenia.
Three of 6 reviewed studies showed that modafinil may improve short-term memory,
attention, and the ability to shift mental sets. Two neuroimaging studies
identified functional correlates in areas associated with working memory
functions. The main adverse effect was found to be a small risk of psychosis
exacerbation, which was seen in 5 of 83 patients (6.0%) in the active treatment
groups as compared to 2 of 70 patients (2.9%) in the placebo groups.
CONCLUSIONS: While the available data suggest that modafinil is generally well
tolerated and may have some efficacy in the treatment of antipsychotic-induced
sedation and cognitive domains, the small sample sizes, contradictory results,
and methodological differences between trials, especially with respect to
cognitive testing, make it difficult to draw firm conclusions about the overall
effectiveness of modafinil as an adjunct in the treatment of schizophrenia.
Well-powered, prospective, randomized placebo-controlled trials using the
MATRICS battery concomitantly with functional outcome measures are necessary to
elucidate modafinil's efficacy and effectiveness as an adjunctive treatment for
sedation, negative symptoms, and cognitive deficits in schizophrenia. Hence,
before prescribing modafinil to a schizophrenia patient, the possible risks and
benefits of each particular case should be evaluated. Limited behavioural repertoire impacts quality of life in chronic schizophrenia.
We have previously shown that the amount of movement exhibited by patients with
schizophrenia is positively correlated with the volume of left anterior
cingulate cortex and that this quantity of movement can be increased by
modafinil. However, increased movement in itself may be of limited clinical
significance. Hence, we sought to analyse the 'structure' of spontaneous
movement in patients with schizophrenia and to examine whether the chunking of
spontaneous activity has a neuroanatomical basis. 'Actiwatches' were used to
record spontaneous motor activity over a 20 hour period in sixteen male patients
with schizophrenia. Time-series data were analysed for the number of discrete
spontaneous activities, which might indicate a degree of structure to ongoing
activity. Subjects underwent a whole-brain structural MRI scan. The 'number of
discrete movement epochs' correlated with volumes of regions within bilateral
rostro-ventral putamen and temporal poles. These data suggest that in people
with schizophrenia the volume of bilateral putamen may influence the complexity
of their behaviours, as distinct from the overall amount of behaviour. The
results are presented in the context of a large body of previous research
examining the role of the basal ganglia in motor and cognitive pattern
generation. BACKGROUND: Patients with schizophrenia often suffer from cognitive deficits and
negative symptoms that are poorly responsive to antipsychotics including
clozapine. Clozapine-induced sedation can worsen cognition and impair social and
occupational functioning.
OBJECTIVES: To evaluate the efficacy, tolerability, and safety of modafinil for
negative symptoms, cognition, and wakefulness/fatigue in DSM-IV-diagnosed
schizophrenia patients treated with clozapine.
METHOD: A double-blind, placebo-controlled, flexible-dosed 8-week pilot trial
was conducted between September 2003 and September 2007, adding modafinil up to
300 mg/d to stabilized schizophrenia outpatients receiving clozapine.
Psychopathology, cognition, and wakefulness/fatigue were assessed with standard
rating scales.
RESULTS: Thirty-five patients were randomly assigned to treatment with study
drug and included in the analysis. Modafinil did not reduce negative symptoms or
wakefulness/fatigue or improve cognition compared to placebo. Modafinil was well
tolerated and did not worsen psychosis.
CONCLUSIONS: Results of this pilot trial do not support routine use of modafinil
to treat negative symptoms, cognitive deficits, or wakefulness/fatigue in
patients on clozapine. However, given our limited power to detect a treatment
effect and the clear possibility of a type II error, larger trials are needed to
resolve or refute a potential therapeutic effect of uncertain magnitude.
TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00573417. Modafinil improves working memory in healthy subjects and individuals diagnosed
with schizophrenia and Attention Deficit/Hyperactivity Disorder, though the
effects of modafinil have not been evaluated on working memory in
methamphetamine-dependent subjects. This double-blind, placebo-controlled study
evaluated whether a daily dose of 400 mg of modafinil, administered over three
consecutive days, would enhance performance on a measure of working memory
relative to test performance at baseline and following 3 days of placebo
administration in 11 methamphetamine addicted, nontreatment-seeking volunteers.
The results revealed that participants demonstrating relatively poor performance
on the third day of a 3-day washout period (ie, at baseline), showed significant
improvement on measures of working memory, but not on measures of episodic
memory or information processing speed. In contrast, for participants
demonstrating relatively high performance at baseline, modafinil administration
did not affect test scores. The findings provide an initial indication that
modafinil can reverse methamphetamine-associated impairments in working memory. BACKGROUND: Emotional impairments are important determits of functional
outcome in psychosis, and current treatments are not particularly effective.
Modafinil is a wake-promoting drug that has been shown to improve emotion
discrimination in healthy individuals and attention and executive function in
schizophrenia. We aimed to establish whether modafinil might have a role in the
adjuvant treatment of emotional impairments in the first episode of psychosis,
when therapeutic endeavor is arguably most vital.
METHODS: Forty patients with a first episode of psychosis participated in a
randomized, double-blind, placebo-controlled crossover design study testing the
effects of a single dose of 200 mg modafinil on neuropsychological performance.
Emotional functions were evaluated with the emotional face recognition test, the
affective go-no go task, and the reward and punishment learning test. Visual
analogue scales were used throughout the study to assess subjective mood
changes.
RESULTS: Modafinil significantly improved the recognition of sad facial
expressions (z = 2.98, p = .003). In contrast, there was no effect of modafinil
on subjective mood ratings, on tasks measuring emotional sensitivity to reward
or punishment, or on interference of emotional valence on cognitive function, as
measured by the affective go-no go task.
CONCLUSIONS: Modafinil improves the analysis of emotional face expressions. This
might enhance social function in people with a first episode of psychosis. Modafinil (2-((diphenylmethyl)sulfinyl)acetamide) is described as an atypical
stimulant and is a putative cognition enhancer for schizophrenia, but the
precise mechanisms of action remain unclear. Receptor knockout (KO) mice offer
an opportunity to identify receptors that contribute to a drug-induced effect.
Here we examined the effects of modafinil on exploration in C57BL/6J mice, in
dopamine drd1, drd2, drd3, and drd4 wild-type (WT), heterozygous (HT), and KO
mice, and in 129/SJ mice pretreated with the drd1 antagonist SCH23390 using a
cross-species test paradigm based on the behavioral pattern monitor. Modafinil
increased activity, specific exploration (rearing), and the smoothness of
locomotor paths (reduced spatial d) in C57BL/6J and 129/SJ mice (increased
holepoking was also observed in these mice). These behavioral profiles are
similar to that produced by the dopamine transporter inhibitor GBR12909.
Modafinil was ineffective at increasing activity in male drd1 KOs, rearing in
female drd1 KOs, or reducing spatial d in all drd1 KOs, but produced similar
effects in drd1 WT and HT mice as in C57BL/6J mice. Neither dopamine drd2 nor
drd3 mutants attenuated modafinil-induced effects. Drd4 mutants exhibited a
genotype dose-dependent attenuation of modafinil-induced increases in specific
exploration. Furthermore, the drd1 KO effects were largely supported by the
SCH23390 study. Thus, the dopamine drd1 receptor appears to exert a primary role
in modafinil-induced effects on spontaneous exploration, whereas the dopamine
drd4 receptor appears to be important for specific exploration. The
modafinil-induced alterations in exploratory behavior may reflect increased
synaptic dopamine and secondary actions mediated by dopamine drd1 and drd4
receptors. RATIONALE: Sedation is a common side effect of clozapine treatment and may
exacerbate metabolic consequences of poor diet and exercise habits that are
common in patients with schizophrenia. Modafinil has been proposed as a
treatment for clozapine-induced sedation and metabolic abnormalities.
OBJECTIVE: To estimate the effect sizes and person-to-person variation in
anthropometric measures, glucose and lipid metabolism, and diet on modafinil
treatment for future randomized control trials.
METHODS: A double-blind, placebo-controlled, flexible-dosed 8-week pilot trial
was conducted, adding modafinil up to 300 mg/day to stabilized schizophrenia
outpatients receiving clozapine. Blood pressure, weight, BMI, laboratory assays,
and dietary intake were tracked to monitor changes in metabolic markers.
RESULTS: Thirty-five participants were randomly assigned to treatment with study
drug or placebo and were included in the analysis. Modafinil did not improve
blood pressure, weight, BMI, glucose or lipid metabolism compared to placebo.
Modafinil was well tolerated and did not worsen psychosis.
CONCLUSIONS: Results of this pilot trial do not support routine use of modafinil
to counteract increased weight and metabolic diseases in patients taking
clozapine. However, the effects of modafinil on weight and insulin regulation
warrant further investigation with effect sizes of 0.4 to 0.6. RATIONALE: Cognitive impairments are important determits of functional
outcome in psychosis, which are inadequately treated by antipsychotic
medication. Modafinil is a wake-promoting drug that has been shown to improve
attention, memory and executive function in the healthy population and in
patients with schizophrenia.
OBJECTIVES: We aimed to establish modafinil's role in the adjunctive treatment
of cognitive impairments in the first episode of psychosis, a time when symptoms
may be more malleable than at chronic stages of the disease.
METHODS: Forty patients with a first episode of psychosis participated in a
randomised, double-blind, placebo-controlled crossover design study assessing
the effects of a single dose of 200 mg modafinil on measures of executive
functioning, memory, learning, impulsivity and attention.
RESULTS: Modafinil improved verbal working memory (d = 0.24, p = 0.04), spatial
working memory errors (d = 0.30, p = 0.0004) and strategy use (d = 0.23,
p = 0.03). It also reduced discrimination errors in a task testing impulsivity.
Modafinil showed no effect on impulsivity measures, sustained attention,
attentional set-shifting, learning or fluency.
CONCLUSIONS: Modafinil selectively enhances working memory in first episode
psychosis patients, which could have downstream effects on patients' social and
occupational functioning. RATIONAL: In recent years, evidence suggests that modafinil may be useful for
certain symptom domains of schizophrenia, especially for the negative and
cognitive symptoms. However, the results are not consistent.
OBJECTIVE: This study was designed to investigate the effect of modafinil added
to risperidone in patients with chronic schizophrenia in a double blind and
randomized clinical trial.
METHODS: Participants were inpatients males (35) and females (11), ages 20-49
years at two teaching psychiatric hospital in Iran. All patients were in the
active phase of the illness and met DSM-IV-TR criteria for schizophrenia.
Patients were allocated in a random fashion 23 patients to risperidone 6 mg/day
plus modafinil 200 mg/day and 23 patients to risperidone 6 mg/day plus placebo.
The principal measure of outcome was the positive and negative syndrome scale
(PANSS). Patients were assessed by a psychiatrist at baseline and after 2, 4, 6
and 8 weeks after the start of medication.
RESULTS: The modafinil group had significantly greater improvement in the
negative symptoms as well as PANSS total scores over the 8-week trial. Therapy
with 200 mg/day of modafinil was well tolerated and no clinically important side
effects were observed.
CONCLUSION: The present study indicates modafinil as a potential adjunctive
treatment strategy for treatment of schizophrenia particularly the negative
symptoms. Nevertheless, results of larger-controlled trials are needed before
recommendation for broad clinical application can be made. BACKGROUND: Modafinil, a putative cognitive enhancing drug, has previously been
shown to improve performance of healthy volunteers as well as patients with
attention deficit disorder and schizophrenia, mainly in tests of executive
functions. The aim of this study was to investigate the effects of modafinil on
non-verbal cognitive functions in healthy volunteers, with a particular focus on
variations of cognitive load, measures of motivational factors and the effects
on creative problem-solving.
METHODS: A double-blind placebo-controlled parallel design study evaluated the
effect of 200 mg of modafinil (N = 32) or placebo (N = 32) in non-sleep deprived
healthy volunteers. Non-verbal tests of divergent and convergent thinking were
used to measure creativity. A new measure of task motivation was used, together
with more levels of difficulty on neuropsychological tests from the CANTAB
battery.
RESULTS: Improvements under modafinil were seen on spatial working memory,
planning and decision making at the most difficult levels, as well as visual
pattern recognition memory following delay. Subjective ratings of enjoyment of
task performance were significantly greater under modafinil compared with
placebo, but mood ratings overall were not affected. The effects of modafinil on
creativity were inconsistent and did not reach statistical significance.
CONCLUSIONS: Modafinil reliably enhanced task enjoyment and performance on
several cognitive tests of planning and working memory, but did not improve
paired associates learning. The findings confirm that modafinil can enhance
aspects of highly demanding cognitive performance in non-sleep deprived
individuals. This article is part of a Special Issue entitled 'Cognitive
Enhancers'. Modafinil is a central nervous system wake promoting agent used for the
treatment of excessive daytime sleeping. Its vigilance promoting properties and
low abuse potential has intrigued the scientific community and has led to use it
as a cognitive enhancer, before its neural functions were understood. Here, we
review the effects of modafinil in human cognition and emotion and its specific
actions on symptoms in patients with schizophrenia and whether these are
consistently effective throughout the literature. We also performed a systematic
review on the effects of modafinil on neurotransmitter signalling in different
areas of the brain in order to better understand the neuromechanisms of its
cognitive and emotional enhancing properties. A review of its effects in
schizophrenia suggests that modafinil facilitates cognitive functions, with
pro-mnemonic effects and problem solving improvements. Emotional processing also
appears to be enhanced by the drug, although to date there are only a limited
number of studies. The systematic review on the neurochemical modulation of the
modafinil suggests that its mnemonic enhancing properties might be the result of
glutamatergic and dopaminergic increased neuronal activation in the hippocampus
and in the prefrontal cortex respectively. Other neurotransmitters were also
activated by modafinil in various limbic brain areas, suggesting that the drug
acts on these brain regions to influence emotional responses. These reviews seek
to delineate the neuronal mechanisms by which modafinil affects cognitive and
emotional function. This article is part of a Special Issue entitled 'Cognitive
Enhancers'. OBJECTIVE: To examine the efficacy and safety of modafinil on parkinsonism and
excessive daytime sleepiness (EDS), as well as on negative symptoms and
cognitive abilities in patients with schizophrenia or schizoaffective disorder
(DSM-IV criteria) in a randomized double-blind placebo-controlled 8-week study.
METHODS: Twenty-four male patients, who were aged 20-63 years and on stable dose
of second generation antipsychotic medications and with a negative symptom score
of ≥ 20 on the Positive and Negative Syndrome Scale (PANSS), were randomized
into either the modafinil (n=12) or placebo (n=12) group. The modafinil group
received flexible does of modafinil 50-200mg/day. Primary measurements were the
Simpson-Angus Scale (SAS) for extrapyramidal side effects (EPS), the Epworth
Sleepiness Scale (ESS), the PANSS and a neuropsychological (NP) test battery.
Data were collected on Days 0, 14, 28, 42 and 56 for rating scales, and on Days
0, 28 and 56 for NP tests.
RESULTS: Mixed model analyses showed a significant group-x-time interaction for
total SAS scores (P<0.006), with scores decreasing in the modafinil group but
remaining the same in the placebo group. There were no significant group-x-time
interactions for scores of ESS (total), PANSS (total, positive and negative),
and NP tests (composite and domains) (all P's>0.5). No significant adverse
events were observed.
CONCLUSION: The data suggest that modafinil was a safe adjunctive treatment
which improved parkinsonian symptoms and signs in patients with schizophrenia or
schizoaffective disorder. Further studies in larger samples and with longer
study time are needed to test/confirm the beneficial effects of modafinil on
motor function. Control-related cognitive processes such as rule selection are associated with
cortical oscillations in the theta, alpha and, beta ranges, and modulated by
catecholamine neurotransmission. Thus, a potential strategy for improving
cognitive control deficits in schizophrenia would be to use pro-catecholamine
pharmacological agents to augment these control-related oscillations. In a
double-blind, placebo-controlled (within-subjects) study, we tested the effects
of adjunctive single-dose modafinil 200 mg on rule-related 4-30 Hz oscillations
in 23 stable schizophrenia patients, using EEG during cognitive control task
performance. EEG data underwent time-frequency decomposition with Morlet
wavelets to determine the power of 4-30 Hz oscillations. Modafinil (relative to
placebo) enhanced oscillatory power associated with high-control rule selection
in theta, alpha, and beta ranges, with modest effects during rule maintece.
Modafinil treatment in schizophrenia augments middle-frequency cortical
oscillatory power associated with rule selection, and may subserve diverse
subcomponent processes in proactive cognitive control. We conducted a systematic review and meta-analysis of randomized controlled
trials (RCTs) of modafinil or armodafinil (ar/mod) augmentation in
schizophrenia. We searched PubMed, clinical trial registries, reference lists,
and other sources for parallel group, placebo-controlled RCTs. Our primary
outcome variable was the effect of ar/mod on negative symptom outcomes. Eight
RCTs (pooled N = 372; median duration, 8 weeks) met our selection criteria.
Ar/mod (200 mg/day) significantly attenuated negative symptom ratings (6 RCTs; N
= 322; standardized mean difference [SMD], -0.26; 95% CI, -0.48 to -0.04). This
finding remained similar in all but one sensitivity analysis - when the only RCT
in acutely ill patients was excluded, the outcome was no longer statistically
significant (SMD, -0.17; 95% CI, -0.51 to 0.06). The absolute advantage for
ar/mod was small: just 0.27 points on the PANSS-N (6 RCTs). Ar/mod attenuated
total psychopathology ratings (7 RCTs; N = 342; SMD, -0.23; 95% CI, -0.45 to
-0.02) but did not influence positive symptom ratings (5 RCTs; N = 302; mean
difference, -0.58; 95% CI, -1.71 to 0.55). Although data were limited,
cognition, fatigue, daytime drowsiness, adverse events, and drop out rates did
not differ significantly between ar/mod and placebo groups. Fixed and random
effects models yielded similar results. There was no heterogeneity in all but
one analysis. Publication bias could not be tested. We conclude that ar/mod (200
mg/day) is safe and well tolerated in the short-term treatment of schizophrenia.
Ar/mod reduces negative symptoms with a small effect size; the absolute
advantage is also small, and the advantage disappears when chronically ill
patients or those with high negative symptom burden are treated. Ar/mod does not
benefit or worsen other symptom dimensions in schizophrenia. |
What is the role of the Ada O6-alkylguanine alkyltransferase in bacteria? | The Ada O6-methylguanine-DNA methyltransferase is a multifunctional protein, product of the ada gene. Ada functions in DNA repair by direct dealkylation of alkylated DNA lesions, such as the toxic, mutagenic and carcinogenic O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) which are restored to guanine and thymine. Ada accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue. When methylated at Cys-69, Ada becomes a transcriptional activator of the genes in the ada regulon, including its own. The ada gene controls the inducible resistance to alkylation mutagenesis and killing (the adaptive response). Ada alkyltransferase (ATase) is induced by exposure to low doses of methylating agents. During exponential growth, Ada removes lesions responsible for G:C to A:T transitions and G:C to C:G transversions, while in stationary populations it removes lesions causing G:C to A:T and A:T to G:C transitions, and G:C to C:G, A:T to C:G, and A:T to T:A transversions. Thus, Ada protein acts both as a positive regulator of the ada response and as a DNA repair enzyme. | The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues
positioned identically with respect to cysteines in the E. coli ada
O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these
residues were each substituted by a glycine to generate altered forms of the ogt
protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of
homology, eliminated functional activity confirming that this residue is the
methyl-accepting cysteine in the active site of the protein. Substitution of
cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein
activity demonstrating that this cysteine is not directly involved with the
transfer of O6-methylguanine adducts. The inactivation of human and Escherichia coli O6-alkylguanine-DNA
alkyltransferase by O6-methylguanine and O6-benzylguanine was compared. When
HT29 cell extracts or E. coli Ada protein were incubated in the presence of 200
microM O6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and
39% of control levels respectively. However, under the same conditions
O6-benzylguanine completely depleted alkyltransferase activity in the extract
from human cells but had virtually no effect on the Ada protein. Incubation of
the HT29 cell alkyltransferase with O6-benzyl[3H]guanine resulted in a
time-dependent production of [3H]guanine. No similar production of [3H]guanine
was observed in the presence of the Ada protein. In CHO cells transfected with
the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT),
treatment with 500 microM O6-methylguanine inhibited both alkyltransferases by
greater than 85%. In contrast, 2 microM O6-benzylguanine inhibited human
alkyltransferase expressed in CHO-MGMT cells by greater than 99% though
concentrations as high as 25 microM for 24 h had no inhibitory effects on the
bacterial alkyltransferase expressed in CHO-ada cells. This selective inhibition
was also observed in vivo in transgenic mice expressing ada in the liver where
O6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase
activity compared to 95% in non-transgenic mice, consistent with inhibition of
only the mammalian alkyltransferase and maintece of bacterial
alkyltransferase activity in these animals. Thus, while O6-methylguanine at high
concentrations inactivates both bacterial and mammalian alkyltransferases,
O6-benzylguanine is a substrate only for the mammalian protein and is unable,
perhaps due to steric hindrance, to inhibit the Ada protein. A mutant of Bacillus subtilis defective in the constitutive activity of
O6-alkylguanine-DNA alkyltransferase was isolated from a strain (ada-1)
deficient in the adaptive response to DNA alkylation. Cells carrying the
mutation dat-1 which was responsible for the defect in constitutive activity
exhibited hypersensitivity for lethality and mutagenesis when challenged with
methyl-nitroso compounds. The constitutive activity is independent of the
adaptive response, and seems to function as a basal defense against
environmental alkylating agents. The inducible resistance to alkylation mutagenesis and killing in Escherichia
coli (the adaptive response) is controlled by the ada gene. The Ada protein acts
both as a positive regulator of the response and as a DNA repair enzyme,
correcting premutagenic O6-alkylguanine in DNA by suicidal transfer of the alkyl
group to one of its own cysteine residues. We have determined the DNA sequence
of the cloned ada+ gene and its regulatory region. The data reveal potential
sites of ada autoregulation. Amino acid sequence determinations show that the
active center for the O6-methylguanine-DNA methyltransferase is located close to
the polypeptide COOH terminus and has the unusual sequence -Pro-Cys-His-,
preceded by a very hydrophobic region. These same structural features are
present at the active site of thymidylate synthase, suggesting a common chemical
mechanism for activation of the cysteine. Although the human O6-alkylguanine-DNA alkyltransferase (AGT) is very sensitive
to inactivation by O6-benzylguanine (BG) or
2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP), the equivalent
protein formed by the carboxyl terminal domain of the product of the Escherichia
coli ada gene (Ada-C) is unaffected by these inhibitors. This difference is
remarkable in view of the substantial similarity between these proteins (33% of
the residues in the common sequence are identical) and is potentially very
important since these inhibitors are under development as drugs to enhance the
anti-tumor activity of alkylating agents. In order to understand the reason for
the resistance of the Ada-C protein, we have made chimeras between Ada-C and AGT
sequences and mutations in the Ada-C protein, expressed the altered proteins in
an E. coli strain lacking endogenous alkyltransferase activity and tested the
inactivation of the resulting proteins by BG or 5-nitroso-BP. Chimeric
alkyltransferase proteins were made in which the residues on the amino side of
the cysteine acceptor site came from Ada-C and the residues on the carboxyl side
came from AGT and vice versa but these did not show sensitivity to BG suggesting
that resistance is produced by residues in both segments of the protein.
Analysis of the Ada-C mutant proteins revealed two sites for mutations that
confer sensitivity to these inhibitors. One of these was tryptophan-336 and the
other was residues lysine-314 and alanine-316. Thus, when the combined mutations
of A316P/W336A were made in the Ada-C sequence, the protein was sensitive to
inactivation by BG. This A316P/W336A mutant protein was even more sensitive to
5-nitroso-BP and the mutant proteins W336A, K314P/A316P and A316P could also be
inhibited by this drug (in decreasing order of sensitivity) although the control
Ada-C and a mutant R335S were not inhibited. These results provide strong
support for the hypothesis that the resistance of the Ada-C alkyl-transferase is
due to a steric effect limiting access to the active site. Insertion of proline
residues at positions 314 and 316 and removal of the bulky tryptophan residue at
position 336 increases the space available at the active site and permits these
inhibitors to be effective. We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt
gene in the sensitivity of Escherichia coli to the mutagenic effects of the
dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt-
bacteria to those in their isogenic ogt+ parental counterparts. The effects of
the uvrABC excision-repair system, the adaptive response, mucAB and umuDC
mutagenic processing, and glutathione bioactivation on the differential
responses of ogt- and ogt+ bacteria were also studied. Mutation induction was
monitored by measuring the frequency of forward mutations to L-arabinose
resistance. Induced mutations occurred only in excision repair-defective strains
and were totally (with dibromomethane) or substantially (with dibromoethane)
dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased
mutagenic response to both dibromoalkanes was also seen in ogt- bacteria that
overexpressed the ogt protein from a multicopy plasmid, indicating that the
differences in mutability between ogt+ and ogt- bacteria were not dependent on
the ogt- null allele carried by the defective strain. The ATase encoded by the
constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity
than the ada ATase induced by exposure to low doses of a methylating agent. The
mutagenicity promoted by the ogt ATase was dependent on both glutathione
bioactivation and SOS mutagenic processing. To our knowledge, this paper
presents for the first time evidence that DNA ATases, in particular the ATase
encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging
agent. The mechanism of this effect has yet to be established. We have previously reported the isolation of an Escherichia coli K12 mutant that
is extremely sensitive to mutagenesis by low doses of ethylating agents. We now
show by Southern analysis that the mutation involves a gross deletion covering
at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase
activity is present in cell-free extracts of an ada::Tn10 derivative of these
bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was
attributable to ogt and not to any other loci covered by the deletion was
obtained by constructing derivatives. Thus an ogt::kanr disruption mutation was
introduced into the parental ogt+ bacteria, and the ogt::kanr mutation was then
eliminated by cotransduction of ogt+ with the closely linked Tetr marker
(zcj::Tn10). The delta(ogt-fnr) deletion or ogt::kanr disruption mutants were
highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by
the induction of forward mutations to L-arabinose resistance (Arar).
Furthermore, the number of Arar mutants increased linearly with dose, unlike the
case in ogt+ bacteria, which had a threshold dose below which no mutants
accumulated. Differences in mutability were even greater with propyl
methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy
plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt- mutant
strains and also methylmethanesulphonate mutagenesis in ada- bacteria. A sample
of AB1157 obtained from the E. coli K12 genetic stock centre also had a deletion
covering the ogt and fnr genes. Since such deletions greatly influence the
mutagenic responses to alkylating agents, a survey of the presence of the ogt
gene in the E. coli K12 strain being used is advisable. The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to
transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly
restoring these alkylated DNA lesions to guanine and thymine. In addition to
being exquisitely sensitive to the mutagenic effects of methylating agents, E.
coli ada ogt null mutants display a higher spontaneous mutation rate than the
wild type. Here, we determined which base substitution mutations are elevated in
the MTase-deficient cells by monitoring the reversion of six mutated lacZ
alleles that revert via each of the six possible base substitution mutations.
During exponential growth, the spontaneous rate of G:C to A:T transitions and
G:C to C:G transversions was elevated about fourfold in ada ogt double mutant
versus wild-type E. coli. Furthermore, compared with the wild type, stationary
populations of the MTase-deficient E. coli (under lactose selection) displayed
increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively)
and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-,
and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated
spontaneous mutation rates for any base substitution event, and the cloned ada
and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt
MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can
repair the endogenously produced DNA lesions responsible for each of the five
base substitution events that are elevated in MTase-deficient cells. Simple
methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions
in these strains but did not significantly induce G:C to C:G, A:T to C:G, and
A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak
methylating agent) is not the only metabolite responsible for endogenous DNA
alkylation and that at least some of the endogenous metabolites that cause
O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents. O6-Methylguanine is removed from DNA via the transfer of the methyl group to a
cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA
alkyltransferase. The human alkyltransferase is inactivated by the free base
O6-benzylguanine, raising the possibility that substantially larger alkyl groups
could also be accepted as substrates. However, the Escherichia coli
alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C
protein was rendered capable of reaction by the incorporation of two
site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to
alanine (W336A) or glycine (W336G). These changes increase the space at the
active site of the protein where Cys321 is buried and thus permit access of the
O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with
O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong
support for the concept that binding of DNA to the Ada-C protein activates the
protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer
oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the
E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C
alkyltransferases reacted very rapidly with this 16-mer substrate and
preferentially repaired it when incubated with a mixture of the methylated and
benzylated 16-mers. These results show that benzyl groups are better substrates
than methyl groups for alkyltransferases provided that steric factors do not
prevent binding of the substrate in the correct orientation for alkyl group
transfer. O6-Alkylguanine DNA-alkyltransferase (ATase) repairs toxic, mutagenic and
carcinogenic O6-alkylguanine (O6-alkG) lesions in DNA by a highly conserved
reaction involving the stoichiometric transfer of the alkyl group to the active
centre cysteine residue of the ATase protein. In the Escherichia coli Ada ATase,
which is effectively refactory to inhibition by O6-benzylguanine (O6-BzG), the
residue corresponding to glycine-160 (G160) for the mammalian proteins of this
class is replaced by a tryptophan (W). Therefore, to investigate the potential
role of the G160 of the human ATase (hAT) protein in determining sensitivity to
O6-BzG, site-directed mutagenesis was used to produce a mutant protein
(hATG160W) substituted at position 160 with a W residue. The hATG160W mutant was
found to be stably expressed and was 3- and 5-fold more sensitive than hAT to
inactivation by O6-BzG, in the absence and presence of additional calf-thymus
DNA respectively. A similar, DNA dependent increased sensitivity of the hATG160W
mutant relative to wild-type was also found for O6-methylguanine mediated
inactivation. The potential role of the W160 residue in stabilising the binding
of the O6-alkG to the protein is discussed in terms of a homology model of the
structure of hAT. The region occupied by G/W-160 forms the site of a putative
hinge that could be important in the conformational change that is likely to
occur on DNA binding. Three sequence motifs have been identified in this region
which may influence O6-BzG access to the active site; YSGG or YSGGG in mammals
(YAGG in E. coli Ogt, YAGS in Dat from Bacillus subtilis), YRWG in E. coli Ada
and Salmonella typhimurium (but YKWS in Saccharomyces cerevisiae) or YRGGF in
AdaB from B. Subtilis. Finally,conformational and stereoelectronic analysis of
the putative transition states for the alkyl transfer from a series of
inactivators of hAT, including O6-BzG was undertaken to rationalise the
unexpected weak inhibition shown by the alpha-pi-unsaturated electrophiles. The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA
methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric
globular polypeptide with two distinct alkylacceptor activities located in two
domains. The two domains are of nearly equal size and are connected by a hinge
region. The Ada protein accepts stoichiometrically the alkyl group from
O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at
the Cys-69 residue. This protein functions in DNA repair by direct dealkylation
of mutagenic O6-alkylguanine. The protein methylated at Cys-69 becomes a
transcriptional activator of the genes in the ada regulon, including its own.
Each of the two domains functions independently as an alkyl acceptor. The
purified homogeneous protein is unstable at 37 degrees C and spontaneously loses
about 30% of its secondary structure in less than 30 min concomitant with a
complete loss of activity. However, sedimentation equilibrium studies indicated
that the inactive protein remains in the monomeric form without aggregation.
Furthermore, electrospray mass spectroscopic analysis indicated the absence of
oxidation of the inactive protein. This temperature-dependent inactivation of
the Ada protein is inhibited by DNA. In the presence of increasing
concentrations of urea or guanidine, the protein gradually loses more than 80%
of its structure. The two alkyl acceptor activities appear to be differentially
sensitive to unfolding and the phosphotriester methyltransferase activity is
resistant to 7 M urea. The partial or complete unfolding induced by urea or
guanidine is completely reversed within seconds by removal of the denaturant.
The heat-coagulated protein can also be restored to full activity by cycling it
through treatment with 8 M urea or 6 M guanidine. These results suggest that the
nascent or unfolded Ada polypeptide folds to a metastable form which is active
and that the thermodynamically stable structure is partially unfolded and
inactive. Spontaneous mutagenesis in O6-alkylguanine-DNA alkyltransferase-proficient and
-deficient (ada ogt mutants) Escherichia coli was studied in two ways: in
bacteria growing in nonselective liquid medium and in bacteria resting on
selective agar plates. ATase mutants showed similar spontaneous mutation rates
as ATase proficient bacteria during growth phase; an excess of mutants arising
in nondividing cells. The resting-associated mutagenesis in ada + ogt + uvr-
bacteria was biphasic; the high sensitive range being triggered beyond the first
6 days after plating. Contrarily, spontaneous Lacc mutants from ada- ogt- uvr-
cells steadily increased over the 8 day period of plate incubation. These
results suggested that, in the absence of nucleotide excision repair, the repair
by both the Ada and the Ogt ATases is not saturated until the cells have been
resting for 6 days. The spontaneous LacI-d mutation spectrum of ada + ogt + uvr-
bacteria growing in non-selective liquid medium served as a baseline to
determine the mutation events increased in the ATase-deficient derivative upon
prolonged incubation on selective plates. The percentage of G:C-->A:T
transitions, presumably driven by unrepaired O6-alkylguanine lesions, was
increased at the expense of other mutation types. G:C-->A:T transitions
accumulated with a pronounced 5'PuG bias, suggesting that the endogenous
metabolite(s) responsible for this mutation class is an SN1 type alkylating
compound(s). Accordingly, the site distribution of G:C-->A:T transitions in
nondividing ATase defective bacteria showed similarities with the spectra
induced by alkylnitrosoureas, particularly with those generating bulky alkylated
DNA adducts. The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved
in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an
important role in most organisms in attenuating the cytotoxic and mutagenic
effects of certain classes of alkylating agents. A genomic clone encompassing
the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on
the basis of sequence homology with corresponding genes in Saccharomyces
cerevisiae and man. The DmAGT gene is located at position 84A on the third
chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame
encoding 194 amino acids. The MNNG-hypersensitive phenotype of
alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA.
Furthermore, alkyltransferase activity was identified in crude extracts of
Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by
preincubation of the extract with an oligonucleotide containing a single
O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in
contrast to the human alkyltransferase, the Drosophila alkyltransferase is
resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion
assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well
as A:T-->G:C transition mutations in vivo. These results demonstrate the
presence of an alkyltransferase specific for the repair of O 6-methylguanine and
O 4-methylthymine in Drosophila. |
List the neurotransmitters that are metabolized by MAOA. | The monoamine oxidase-A (MAOA) gene plays a vital role in the metabolism of neurotransmitters, e.g, serotonin, norepinephrine, and dopamine. | BACKGROUND: The monoamine oxidase-A (MAOA) gene plays a vital role in the
metabolism of neurotransmitters, e.g, serotonin, norepinephrine, and dopamine. A
polymorphism in the promoter region (MAOA-uVNTR) affects transcriptional
efficiency. Allelic variation in MAOA-uVNTR has been associated with body mass
index (BMI). We extended previous work by examining relations among this
polymorphism and serum lipid levels.
MATERIAL/METHODS: The sample consisted of 74 males enrolled in a study of
caregivers for relatives with dementia. Regression models, adjusted for age,
race, group status (caregiver/control), and cholesterol lowering medication
(yes/no), were used to examine associations between high verses low MAOA-uVNTR
activity alleles and total cholesterol, HDL, LDL, VLDL, LDL/HDL ratio,
triglycerides, and BMI.
RESULTS: Higher total cholesterol (p<0.03), LDL/HDL ratio (p<0.01),
triglycerides (p<0.02), and VLDL (p<0.02) were associated with low activity
MAOA-uVNTR alleles. HDL and LDL were modestly related to MAOA-uVNTR activity,
however, they did not reach the conventional significance level (p<0.07 and
p<0.10, respectively). BMI (p<0.74) was unrelated to MAOA-uVNTR transcription.
CONCLUSIONS: The present findings suggest that MAOA-uVNTR may influence lipid
levels and individuals with less active alleles are at increased health risk. OBJECTIVE: Monoamine oxidase-A (MAO-A) is a key mitochondrial enzyme that
metabolizes biogenic amine neurotransmitters such as dopamine and serotonin.
Individuals with atypical depression (AD) are particularly responsive to
treatment with MAO inhibitors (MAOIs). Biomarker tests are essential for prompt
diagnosis of AD, and to identify those with an altered brain neurotransmitter
metabolism who may selectively respond to MAOI therapy.
METHODS: In a sample of 118 Scandinavian patients with treatment-resistant
depression who are naive to MAOI therapy, we investigated the associations
between a common MAOA functional promoter polymorphism (MAOA-uVNTR),
cerebrospinal fluid (CSF) neurotransmitter metabolites, and AD susceptibility.
The metabolites for dopamine (homovanillic acid, HVA), serotonin
(5-hydroxyindoleacetic acid) and noradrenaline (3-methoxy-4-hydroxyphenylglycol)
were measured in the CSF.
RESULTS: AD was associated with the female sex and a higher HVA in CSF
(P=0.008). The carriers of the MAOA-uVNTR short allele were significantly
overrepresented among women with AD (P=0.005; odds ratio=4.76; 95% confidence
interval=1.5-13.1; statistical power=80.0%). Moreover, the MAOA-uVNTR genotype
significantly influenced the HVA concentration (P=0.01) and showed a strong
trend in relation to 5-hydroxyindoleacetic acid concentration (P=0.057) in
women. The mediational statistical analyses showed the CSF-HVA concentration as
a key driver of the relationship between MAOA-uVNTR genotype and AD.
CONCLUSION: The association of the MAOA-uVNTR with both susceptibility to AD and
dopamine metabolite (HVA) concentration lends further biological plausibility
for high MAO-A enzyme activity as a mechanistic factor for genetic
predisposition to AD through altered dopamine turnover. Our observations provide
new evidence on the in-vivo functional significance of the MAOA-uVNTR short
allele as a high activity variant. Monoamine oxidase membrane enzymes are responsible for the catalytic breakdown
of extra- and intracellular neurotransmitters and are targets for the
development of central nervous system drugs. We analyzed the dynamics of rat
MAOA by performing multiple independent molecular dynamics simulations of
membrane-bound and membrane-free forms to clarify the relationship between the
mechanics of the enzyme and its function, with particular emphasis on the
significance of membrane attachment. Principal component analysis of the
simulation trajectories as well as correlations in the fluctuations of the
residues pointed to the existence of three domains that define the global
dynamics of the protein. Interdomain anticorrelated movements in the
membrane-bound system facilitated the relaxation of interactions between
residues surrounding the substrate cavity and induced conformational changes
which expanded the active site cavity and opened putative pathways for substrate
uptake and product release. Such events were less pronounced in the
membrane-free system due to differences in the nature of the domit modes of
motion. The presence of the lipid environment is suggested to assist in
decoupling the interdomain motions, consistent with the observed reduction in
enzyme activity under membrane-free conditions. Our results are also in
accordance with mutational analysis which shows that modifications of
interdomain hinge residues decrease the activity of rat MAOA in solution. PURPOSE: Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that
degrades neurotransmitters including serotonin and norepinephrine, are commonly
used to treat neurological conditions including depression. Recently, we and
others identified high expression of MAOA in normal basal prostatic epithelium
and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal
secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of
MAOA, clorgyline, induced secretory differentiation in primary cultures of
normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline
inhibited several oncogenic pathways in PCa cells, suggesting clinical value of
MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for
high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP
cells, which were derived from castration-resistant metastatic PCa and express a
high level of MAOA.
METHODS: Growth of VCaP cells in the presence or absence of clorgyline was
evaluated in vitro and in vivo. Gene expression changes in response to
clorgyline were determined by microarray and validated by quantitative real-time
polymerase chain reaction.
RESULTS: Treatment with clorgyline in vitro inhibited growth and altered the
transcriptional pattern of VCaP cells in a manner consistent with the
pro-differentiation and anti-oncogenic effects seen in treated primary PCa
cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in
androgen-independent growth and metastasis, were significantly downregulated.
Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and
induced transcriptome changes similar to those noted in vitro.
CONCLUSION: Our results support the possibility that anti-depressant drugs that
target MAOA might find a new application in treating PCa. Monoamine oxidases (MAO-A and MAO-B) have a key role in the degradation of amine
neurotransmitters, such as dopamine, norepinephrine and serotonin. We identified
an inherited 240 kb deletion on Xp11.3-p11.4, which encompasses both monoamine
oxidase genes but, unlike other published reports, does not affect the adjacent
Norrie disease gene (NDP). The brothers who inherited the deletion, and thus
have no monoamine oxidase function, presented with severe developmental delay,
intermittent hypotonia and stereotypical hand movements. The clinical features
accord with published reports of larger microdeletions and selective MAO-A and
MAO-B deficiencies in humans and mouse models and suggest considerable
functional compensation between MAO-A and MAO-B under normal conditions. Monoamine Oxidase A (MAOA) is a critical enzyme in the catabolism of
monoaminergic neurotransmitters. MAOA transcriptional activity is thought to be
regulated by a well characterized 30 base pair (bp) variable nucleotide repeat
(VNTR) that lies approximately ∼1000 bp upstream of the transcriptional start
site (TSS). However, clinical associations between this VNTR genotype and
behavioral states have been inconsistent. Herein, we describe a second, 10 bp
VNTR that lies ∼1500 bp upstream of the TSS. We provide in vitro and in silico
evidence that this new VNTR region may be more influential in regulating MAOA
transcription than the more proximal VNTR and that methylation of this CpG-rich
VNTR is genotype dependent in females. Finally, we demonstrate that genotype at
this new VNTR interacts significantly with history of child abuse to predict
antisocial personality disorder (ASPD) in women and accounts for variance in
addition to that explained by the prior VNTR. OBJECTIVES: Monoamine oxidase A (MAOA) modulates metabolism of serotonin and
dopamine metabolism, neurotransmitters involved in regulation of appetite and
food intake. The gene coding for MAOA contains a 30-bp tandem repeat (uVNTR)
polymorphism in its promoter region that has been previously identified to be
associated with obesity with mixed findings in the literature. Our goals were to
replicate the population effects of this functional polymorphism on obesity
risk, and to further explore gender differences and interaction effects with
negative stressors.
METHODS: Analyses were conducted with data on genotypes, measured weight and
height, and self-reported behavioural characteristics among 1101 Chinese
adolescents 11-15 years old living in Wuhan, China.
RESULTS: Girls with the high-activity allele had significantly lower body mass
index (BMI; β = -0.25 ± 0.98, P = 0.011) compared to those with the low activity
allele. Experience of negative familial stressors (e.g., death or illness of
family members, hit or scolded by parents and increased quarrelling with
parents, parents argued frequently) significantly weakened this protective
genetic effect on BMI (P for interaction = 0.043). Stratified analyses showed a
significant protective genetic effect on BMI only within the stratum of low
stress level (β = -0.44 ± 0.14, P = 0.002). No similar effect was observed among
boys.
CONCLUSIONS: Our findings confirm the genetic effects of MAOA uVNTR polymorphism
on BMI in a Chinese adolescent population and suggest potential genetic
interactions with negative familial stressors. Attention deficit hyperactivity disorder (ADHD) is the most frequently diagnosed
behavioral disorder in children with a high frequency of co-morbid conditions
like conduct disorder (CD) and oppositional defiant disorder (ODD). These traits
are controlled by neurotransmitters like dopamine, serotonin and norepinephrine.
Monoamine oxidase A (MAOA), a mitochondrial enzyme involved in the degradation
of amines, has been reported to be associated with aggression, impulsivity,
depression, and mood changes. We hypothesized that MAOA can have a potential
role in ADHD associated CD/ODD and analyzed 24 markers in a group of
Indo-Caucasoid subjects. ADHD probands and controls (N = 150 each) matched for
ethnicity and gender were recruited following the Diagnostic and Statistical
Manual for Mental Disorders-IV. Appropriate scales were used for measuring CD
and ODD traits. Markers were genotyped by PCR-based methods and data obtained
analyzed using the Cocaphase program under UNPHASED. Only eight markers were
found to be polymorphic. rs6323 "G" allele showed higher frequencies in ADHD (P
= 0.0023), ADHD + CD (P = 0.03) and ADHD + ODD (P = 0.01) as compared to
controls. Haplotype analysis revealed statistically significant difference for
three haplotypes in ADHD cases (P < 0.02). Statistically significant differences
were also noticed for haplotypes in ADHD + CD and ADHD + ODD cases (P < 0.01).
LD analysis showed significant variation in different groups.
Multidimensionality reduction analysis showed independent as well as interactive
effects of markers. Genotypes showed correlation with behavioral problems in
ADHD and ADHD + CD. We interpret that MAOA gene variants may contribute to the
etiology of ADHD as well as associated co-morbid CD and ODD in this ethnic
group. BACKGROUND: Anemia during the third trimester of fetal development affects
one-third of the pregcies in the United States and has been associated with
postnatal behavioral outcomes. This study examines how fetal iron deficiency
(ID) interacts with the fetal monoamine oxidase A (MAOA) genotype. MAOA
metabolizes monoamine neurotransmitters. MAOA polymorphisms in humans affect
temperament and modify the influence of early adverse environments on later
behavior.
OBJECTIVE: The aim of the study was to advance translation of developmental ID
research in animal models by taking into account genetic factors that influence
outcomes in human populations.
METHODS: Male infant rhesus monkeys 3-4 mo old born to mothers fed an ID (10 ppm
iron) diet were compared with controls (100 ppm iron). Infant monkeys with high-
or low-transcription rate MAOA polymorphisms were equally distributed between
diet groups. Behavioral responses to a series of structured experiences were
recorded during a 25-h separation of the infants from their mothers.
RESULTS: Infant monkeys with low-transcription MAOA polymorphisms more clearly
demonstrated the following ID effects suggested in earlier studies: a 4% smaller
head circumference, a 39% lower cortisol response to social separation, a 129%
longer engagement with novel visual stimuli, and 33% lesser withdrawal in
response to a human intruder. The high MAOA genotype ID monkeys demonstrated
other ID effects: less withdrawal and emotionality after social separation and
lower "fearful" ratings.
CONCLUSION: MAOA × ID interactions support the role of monoamine
neurotransmitters in prenatal ID effects in rhesus monkeys and the potential
involvement of common human polymorphisms in determining the pattern of
neurobehavioral effects produced by inadequate prenatal nutrition. |
What is the effect of enamel matrix derivative on pulp regeneration? | EMD increased the osteogenic potential of hDPCs. The expression levels of osteogenesis-related genes, such as ALP, DSPP, BMP, and OPN were also upregulated. In addition, the expression levels of odontogenesis-related transcription factors Osterix and Runx2 were upregulated. Proliferated pulp tissue partly filled the space initially occupied by EMDgel and isolated masses within the proliferated pulp tissue. | During odontogenesis, amelogenins from the preameloblasts are translocated to
differentiating odontoblasts in the dental papilla, suggesting that amelogenins
may be associated with odontoblast changes during development. In the present
study, we have explored the effects of enamel matrix derivative (EMD) on the
healing of a pulpal wound. Coronal pulp tissue of permanent maxillary premolars
of miniature swine were exposed through buccal class V cavities. The exposed
pulp was capped with EMD. The contralateral teeth served as controls and were
capped with a calcium hydroxide paste (Dycal). The cavities were sealed with
glass-ionomer cement. After 2 and 4 weeks, the histology of the teeth was
analyzed. In the EMD-treated teeth, large amounts of newly formed dentin-like
hard tissue with associated formative cells outlined the pulpal wound separating
the cavity area from the remaining pulp tissue. Inflammatory cells were present
in the wound area but not subjacent to the newly formed hard tissue.
Morphometric analysis showed that the amount of hard tissue formed in
EMD-treated teeth was more than twice that of the calcium-hydroxide-treated
control teeth (p < 0.001), suggesting that EMD is capable of promoting
reparative processes in the wounded pulp more strongly than is calcium
hydroxide. AIM: To characterize the hard tissue formed in human teeth experimentally pulp
capped either with calcium hydroxide or with Emdogain Gel (Biora AB, Malmö,
Sweden) - a derivative of enamel matrix (EMD), using two markers for dentine;
dentine sialoprotein (DSP) and type 1 collagen (Col I).
METHODOLOGY: Affinity-purified rabbit anti-Col I and anti-DSP polyclonal
antibodies were used to stain histological sections from nine pairs of
contra-lateral premolars that had been experimentally pulp amputated and
randomly capped with EMDgel or calcium hydroxide. Twelve weeks after the teeth
had been pulp capped, they were extracted, fixed, demineralized and serially
sectioned prior to immunohistochemical staining.
RESULTS: In the calcium hydroxide treated teeth DSP was seen in the new hard
tissue which formed a bridge. DSP was also seen in the newly formed hard tissue
in the EMDgel-treated teeth. Proliferated pulp tissue partly filled the space
initially occupied by EMDgel and DSP-stained hard tissue was observed alongside
exposed dentine surfaces as well as in isolated masses within the proliferated
pulp tissue, although the new hard tissue did not cover the pulp exposure. DSP
staining was also seen in the cells lining the hard tissue in both groups. Col I
staining was seen in the newly formed hard tissue in both groups.
CONCLUSIONS: The new hard tissue formed after pulp capping with EMDgel or
calcium hydroxide contained DSP and Col I, considered to be markers for dentine.
Thus, the newly formed hard tissue can be characterized as dentine rather than
unspecific hard tissue. INTRODUCTION: Hydrogels have been widely studied as tissue engineering scaffolds
over the past 2 decades because of their favorable biological properties.
Recently, a new biodegradable glycol chitin-based thermoresponsive hydrogel
scaffold (GC-TRS) was developed that can be easily applied as a mild viscous
solution at room temperature but quickly transforms into a durable hydrogel
under physiological conditions. The aim of this study was to investigate the
effects of GC-TRS on the proliferation and odontogenic differentiation of
colony-forming human dental pulp cells (hDPCs) in the presence of enamel matrix
derivative.
METHODS: Glycol chitin was synthesized by N-acetylation of glycol chitosan. The
morphology of the thermoresponsive hydrogel scaffold was observed by using
scanning electron microscopy. The sol gel phase transition of the aqueous
solution of glycol chitin was investigated by using the tilting method and
rheometer studies. hDPCs were isolated based on their ability to generate
clonogenic adherent cell clusters. The effect of GC-TRS and collagen on cell
viability was examined by performing
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Expression of markers for odontogenic/osteogenic differentiation (ie, dentin
sialophosphoprotein, dentin matrix protein-1, osteonectin, and osteopontin) was
analyzed by performing real-time polymerase chain reaction.
RESULTS: GC-TRS exhibited a highly macroporous and well-interconnected porous
structure. The polymer solution existed in a mildly viscous sol state, but it
transitioned to a gel state and did not flow above approximately 37°C. Rheometer
studies showed that the glycol chitin solution exhibited a fast sol gel
transition approximately at body temperature. GC-TRS and collagen did not
inhibit cell viability until 7 days. Dentin sialophosphoprotein and dentin
matrix protein-1 were expressed by cells cultured in GC-TRS at a higher level
than that in cells cultured in collagen (P < .05). In both the scaffold groups,
dentin sialophosphoprotein, dentin matrix protein-1, and osteopontin messenger
RNA was up-regulated significantly in EMD-treated hDPCs when compared with the
nontreated cells (P < .05).
CONCLUSIONS: GC-TRS allowed the proliferation and odontogenic differentiation of
hDPCs. Furthermore, the differentiation was facilitated by EMD. These results
suggest that GC-TRS has the potential to be used in tissue engineering
techniques for dentin regeneration. The aim was to review the efficacy of the enamel matrix derivative (EMD) in
direct pulp capping (DPC) procedures. Databases were explored using the
following keywords: 'dental', 'dentine', 'enamel matrix derivative', 'pulp
capping' and 'treatment'. The inclusion criteria were: (i) original studies;
(ii) human and animal studies; (iii) reference list of potentially relevant
original and review articles; (iv) intervention: effect of EMD on pulp-capping
procedures; and (v) articles published only in English. Eight studies (four
human and four animal) were included. Among the human studies, two studies
reported that EMD is a more efficient DPC procedure compared with calcium
hydroxide (Ca(OH)2 ). One study reported Ca(OH)2 to be more efficient for DPC
than EMD. One study reported no difference in the efficacies between EMD and
Ca(OH)2 for DPC. All animal studies reported EMD to be more effective in
reparative dentine formation in comparison with Ca(OH)2 . EMD can provide
favourable results in DPC procedures. |
Is depression associated with poor prognosis of brain tumor patients? | Yes. In brain tumor patients depression is associated with shorter survival and worse functional outcomes. | OBJECTIVE: The adverse impact of depression in relation to survival among cancer
patients is currently a subject of great interest in research. In a 5-year
follow-up study, we investigated the association of depression with survival of
patients with a primary brain tumor.
METHODS: The study population consisted of 75 patients with a solitary primary
brain tumor treated surgically at the Oulu Clinic for Neurosurgery, Oulu
University Hospital, in Northern Finland. The patients were interviewed during
admission to the hospital for the tumor surgery. Assessment of depression was
made using the Beck Depression Inventory and the Crown-Crisp Experiential Index.
Information on all deaths within 60 months after tumor operation was collected
from the Cause of Death Register, provided by Statistics Finland.
RESULTS: The patients with a high-grade glioma had a survival time of 22.5
months (standard deviation, 21.4 mo), whereas the corresponding time was 50.2
months (standard deviation, 19.9 mo) for patients with a low-grade glioma and
58.2 months (standard deviation, 9.4 mo) for the patients with a histologically
benign tumor (P < 0.001, difference between groups, Kruskal-Wallis test). In the
subgroup of patients with low-grade gliomas, depressive patients had a
significantly shorter survival time compared with nondepressive subjects (P =
0.031, Kaplan-Meier survival analysis). A corresponding difference was not found
in patients with high-grade gliomas or benign tumors. Tumor location in one
hemisphere compared with bilateral location and wider extent of tumor surgery
was associated with better survival in patients with low-grade gliomas and
benign tumors but not in patients with high-grade gliomas.
CONCLUSION: Preoperative depression seemed to be a significant prognostic factor
for worse survival in low-grade glioma patients. In clinical practice, an
evaluation of depression among brain tumor patients by structured and
standardized diagnostic methods is needed to distinguish the patients whose
depression actually needs treatment. The effective treatment of clinical
depression among brain tumor patients and the impact of treatment on the
patients' chances of survival should be a focus of future research. OBJECT: The authors analyzed changes in depression and contemporary functional
states by using valid tools in a population-based study sample during a 1-year
follow-up period.
METHODS: The study population consisted of 77 patients with a solitary primary
brain tumor treated surgically at the Oulu Clinic for Neurosurgery. Each
patient's depressive status, according to the Beck Depression Inventory (BDI),
and functional outcome, based on the Karnofsky Performance Scale (KPS), were
evaluated before the tumor was surgically treated as well as 3 months and 1 year
after surgery. Before surgery 27 patients (35%) had BDI scores indicating the
presence of depression. These scores were significantly higher in patients with
a history of depression (p = 0.017) and in those with a lower functional outcome
(p = 0.015). In the entire study sample the severity of depression decreased
statistically significantly (p = 0.031) at 3 months postsurgery. A lower
functional status (KPS score < or = 70) in patients was significantly associated
with high depression scores at the 3-month (p = 0.000) and 1-year (p = 0.005)
assessments. The decrease in the level of depression was significant in patients
with an anterior tumor (p = 0.049) and those with a pituitary adenoma (p =
0.019).
CONCLUSIONS: Affective disorders among patients with brain tumors must be
considered immediately after surgery, especially in persons with a depression
history and in those with a coincident physical disability. OBJECTIVES: To assess the long-term survival of brain tumor patients, and in
particular to evaluate the relation of quality of life (QOL) to survival among
low-grade glioma patients.
METHODS: The postoperative survival of 101 brain tumor patients was followed
from surgery (1990-1992) until the end of the year 2003. Depression was
evaluated by the Beck Depression Inventory (BDI) and QOL with Sintonen's 15D
scale before operation and at one year as well as at five years after operation.
RESULTS: The mean survival times in years (SD) were significantly related to
tumor maligcy, being the shortest, 1.9 (0.6), for patients with high-grade
gliomas, while patients with low-grade gliomas or a benign brain tumor had mean
survival times of 9.1 (1.0) and 11.6 (0.5), respectively. At all follow-ups,
depressed low-grade glioma patients had a significantly shorter survival time,
3.3-5.8 years, compared to non-depressed low-grade glioma patients, 10.0-11.7
years. A decreased level of QOL in low-grade glioma patients was significantly
related to the shorter survival.
CONCLUSIONS: The results suggest that depression and decreased QOL among
low-grade glioma patients is related to shorter survival at long-term follow-up.
Decreased QOL may serve as an indicator for poor prognosis in low-grade glioma
patients. |
Super-SILAC is a method used in quantitative proteomics. What is the super-SILAC mix?
(SILAC: Stable Isotopic labelling by aminoacids in cell culture) | The Super-SILAC mix consists of the combination of multiple SILAC-labeled cell lines. | We describe a method to accurately quantify human tumor proteomes by combining a
mixture of five stable-isotope labeling by amino acids in cell culture
(SILAC)-labeled cell lines with human carcinoma tissue. This generated hundreds
of thousands of isotopically labeled peptides in appropriate amounts to serve as
internal standards for mass spectrometry-based analysis. By decoupling the
labeling from the measurement, this super-SILAC method broadens the scope of
SILAC-based proteomics. Mass spectrometry (MS)-based proteomics is increasingly applied in a
quantitative format, often based on labeling of samples with stable isotopes
that are introduced chemically or metabolically. In the stable isotope labeling
by amino acids in cell culture (SILAC) method, two cell populations are cultured
in the presence of heavy or light amino acids (typically lysine and/or
arginine), one of them is subjected to a perturbation, and then both are
combined and processed together. In this study, we describe a different
approach--the use of SILAC as an internal or 'spike-in' standard--wherein SILAC
is only used to produce heavy labeled reference proteins or proteomes. These are
added to the proteomes under investigation after cell lysis and before protein
digestion. The actual experiment is therefore completely decoupled from the
labeling procedure. Spike-in SILAC is very economical, robust and in principle
applicable to all cell- or tissue-based proteomic analyses. Applications range
from absolute quantification of single proteins to the quantification of whole
proteomes. Spike-in SILAC is especially advantageous when analyzing the
proteomes of whole tissues or organisms. The protocol describes the quantitative
analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five
SILAC-labeled cell lines that accurately represents the tissue. It includes the
selection and preparation of the spike-in SILAC standard, the sample preparation
procedure, and analysis and evaluation of the results. Correct classification of cancer patients into subtypes is a prerequisite for
acute diagnosis and effective treatment. Currently this classification relies
mainly on histological assessment, but gene expression analysis by microarrays
has shown great promise. Here we show that high accuracy, quantitative
proteomics can robustly segregate cancer subtypes directly at the level of
expressed proteins. We investigated two histologically indistinguishable
subtypes of diffuse large B-cell lymphoma (DLBCL): activated B-cell-like (ABC)
and germinal-center B-cell-like (GCB) subtypes, by first developing a general
lymphoma stable isotope labeling with amino acids in cell culture (SILAC) mix
from heavy stable isotope-labeled cell lines. This super-SILAC mix was combined
with cell lysates from five ABC-DLBCL and five GCB-DLBCL cell lines. Shotgun
proteomic analysis on a linear ion trap Orbitrap mass spectrometer with high
mass accuracy at the MS and MS/MS levels yielded a proteome of more than 7,500
identified proteins. High accuracy of quantification allowed robust separation
of subtypes by principal component analysis. The main contributors to the
classification included proteins known to be differentially expressed between
the subtypes such as the transcription factors IRF4 and SPI1/PU.1, cell surface
markers CD44 and CD27, as well as novel candidates. We extracted a signature of
55 proteins that segregated subtypes and contained proteins connected to
functional differences between the ABC and GCB-DLBCL subtypes, including many
NF-κB-regulated genes. Shortening the analysis time to single-shot analysis
combined with use of the new linear quadrupole Orbitrap analyzer (Q Exactive)
also clearly differentiated between the subtypes. These results show that high
resolution shotgun proteomics combined with super-SILAC-based quantification is
a promising new technology for tumor characterization and classification. The development of metastasis is a complex, multistep process that remains
poorly defined. To identify proteins involved in the colonization phase of the
metastatic process, we compared the proteome of tumors derived from inoculation
of a panel of isogenic human cancer cell lines with different metastatic
capabilities into the mammary fat pad of immunodeficient mice. Using a protein
standard generated by SILAC-labeling, a total of 675 proteins were identified
and 30 were differentially expressed between at least two of the tumors. The
protein standard contained the proteomes of seven cell lines from multiple
histogenic origins and displayed superior features compared to standard
super-SILAC. The expression of some proteins correlated with metastatic
capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate
dehydrogenase A, while the expression of elongation factor tu correlated
inversely to metastatic capabilities. The expression of these proteins was
biochemically validated, and expression of myosin-9 in clinical breast cancer
samples was further shown to be altered in primary tumors versus corresponding
lymph node metastasis. Our study demonstrates an improved strategy for
quantitative comparison of an unlimited number of tumor tissues, and provides
novel insights into key proteins associated with the colonization phase of
metastasis formation. Cells secrete a large number of proteins to communicate with their surroundings.
Furthermore, plasma membrane proteins and intracellular proteins can be released
into the extracellular space by regulated or non-regulated processes. Here, we
profiled the supernatant of 11 cell lines that are representative of different
stages of breast cancer development by specifically capturing N-glycosylated
peptides using the N-glyco FASP technology. For accurate quantification we
developed a super-SILAC mix from several labeled breast cancer cell lines and
used it as an internal standard for all samples. In total, 1398 unique
N-glycosylation sites were identified and quantified. Enriching for
N-glycosylated peptides focused the analysis on classically secreted and
membrane proteins. N-glycosylated secretome profiles correctly clustered the
different cell lines to their respective cancer stage, suggesting that
biologically relevant differences were detected. Five different profiles of
glycoprotein dynamics during cancer development were detected, and they
contained several proteins with known roles in breast cancer. We then used the
super-SILAC mix in plasma, which led to the quantification of a large number of
the previously identified N-glycopeptides in this important body fluid. The
combination of quantifying the secretome of cancer cell lines and of human
plasma with a super-SILAC approach appears to be a promising new approach for
finding markers of disease. |
What is an acceptable sequence coverage(depth) required for human whole-exome sequencing? | A medium depth may be considered as 8x while the most common values vary between 30x and 60x. Values more than 75x or even up to 125x may be considered for the investigation of rare disease variants. | Hepatocellular carcinoma, one of the most common virus-associated cancers, is
the third most frequent cause of cancer-related death worldwide. By massively
parallel sequencing of a primary hepatitis C virus-positive hepatocellular
carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same
individual, we identified more than 11,000 somatic substitutions of the tumor
genome that showed predomice of T>C/A>G transition and a decrease of the T>C
substitution on the transcribed strand, suggesting preferential DNA repair. Gene
annotation enrichment analysis of 63 validated non-synonymous substitutions
revealed enrichment of phosphoproteins. We further validated 22 chromosomal
rearrangements, generating four fusion transcripts that had altered
transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome
sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense
substitution in a subpopulation of the tumor cells. This first high-resolution
characterization of a virus-associated cancer genome identified previously
uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion
genes, as well as genetic heterogeneity within the tumor. Linkage testing using Affymetrix 6.0 SNP Arrays mapped the disease locus in
TCD-G, an Irish family with autosomal domit retinitis pigmentosa (adRP), to
an 8.8 Mb region on 1p31. Of 50 known genes in the region, 11 candidates,
including RPE65 and PDE4B, were sequenced using di-deoxy capillary
electrophoresis. Simultaneously, a subset of family members was analyzed using
Agilent SureSelect All Exome capture, followed by sequencing on an Illumina
GAIIx platform. Candidate gene and exome sequencing resulted in the
identification of an Asp477Gly mutation in exon 13 of the RPE65 gene tracking
with the disease in TCD-G. All coding exons of genes not sequenced to sufficient
depth by next generation sequencing were sequenced by di-deoxy sequencing. No
other potential disease-causing variants were found to segregate with disease in
TCD-G. The Asp477Gly mutation was not present in Irish controls, but was found
in a second Irish family provisionally diagnosed with choroideremia, bringing
the combined maximum two-point LOD score to 5.3. Mutations in RPE65 are a known
cause of recessive Leber congenital amaurosis (LCA) and recessive RP, but no
domit mutations have been reported. Protein modeling suggests that the
Asp477Gly mutation may destabilize protein folding, and mutant RPE65 protein
migrates marginally faster on SDS-PAGE, compared with wild type. Gene therapy
for LCA patients with RPE65 mutations has shown great promise, raising the
possibility of related therapies for domit-acting mutations in this gene. BACKGROUND: The creation of lymphoblastoid cell lines (LCLs) through
Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a
valuable biomaterial for cell biology research and a renewable source of DNA.
While LCLs have been used extensively in cellular and genetic studies, the
process of cell transformation and expansion during culturing may introduce
genomic changes that may impact their use and the interpretation of subsequent
genetic findings.
RESULTS: We performed whole exome sequencing on a tetrad family using DNA
derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each
individual. We generated over 4.7 GB of mappable sequence to a 125X read
coverage per sample. An average of 19,354 genetic variants were identified.
Comparison of the two DNA sources from each individual showed an average
concordance rate of 95.69%. By lowering the variant calling parameters, the
concordance rate between the paired samples increased to 99.82%. Sanger
sequencing of a subset of the remaining discordant variants did confirm the
presence of de novo mutations arising in LCLs.
CONCLUSIONS: By varying software stringency parameters, we identified 99%
concordance between DNA sequences derived from the two different sources from
the same donors. These results suggest that LCLs are an appropriate
representation of the genetic material of the donor and suggest that EBV
transformation can result in low-level generation of de novo mutations.
Therefore, use of PBMC or early passage EBV-transformed cells is recommended.
These findings have broad-reaching implications, as there are thousands of LCLs
in public biorepositories and individual laboratories. Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for
whole exome sequencing using the Illumina Genome Analyzer (GA) platform. The
protocol involves two-stage library construction. The specificity of exome
enrichment was approximately 80% with 95.6% even coverage of the 34 Mb target
region at an average sequencing depth of 33-fold. Comparison of our results with
whole genome shot-gun resequencing results showed that the exome SNP calls gave
only 0.97% false positive and 6.27% false negative variants. Our protocol is
also well suited for use with whole genome amplified DNA. The results presented
here indicate that there is a promising future for large-scale population
genomics and medical studies using a whole exome sequencing approach. Whole-exome sequencing (Exome-seq) has been successfully applied in several
recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines
and their matched normal samples. We captured 162,073 exons of 16,954 genes and
sequenced the targeted regions to a mean coverage of 56-fold. This study
identified a total of 1517 somatic mutations and validated 934 mutations by
transcriptome sequencing. We detected recurrent mutations in 56 genes. Among
them, 41 have not been described. The mutation rates varied widely among cell
lines. The diversity of the mutation rates was significantly correlated with the
distinct MLH1 copy-number status. Exome-seq revealed intensive genomic
instability in a cell line with MLH1 homozygous deletion, indicated by a
dramatically elevated rate of somatic substitutions, small insertions/deletions
(indels), as well as indels in microsatellites. Notably, we found that MLH1
expression was decreased by nearly half in cell lines with an allelic loss of
MLH1. While these cell lines were negative in conventional microsatellite
instability assay, they showed a 10.5-fold increase in the rate of somatic
indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1
haploinsufficiency in the correction of DNA indel errors. We further analyzed
the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We
observed a much higher rate of indel mutations in the affected cases and
identified recurrent truncating indels in several cancer genes such as VHL,
PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion,
through increasing the rate of indel mutations, could drive the development and
progression of sporadic cancers. Usher syndrome (USH) is a clinically and genetically heterogeneous disorder
characterized by visual and hearing impairments. Clinically, it is subdivided
into three subclasses with nine genes identified so far. In the present study,
we investigated whether the currently available Next Generation Sequencing (NGS)
technologies are already suitable for molecular diagnostics of USH. We analyzed
a total of 12 patients, most of which were negative for previously described
mutations in known USH genes upon primer extension-based microarray genotyping.
We enriched the NGS template either by whole exome capture or by Long-PCR of the
known USH genes. The main NGS sequencing platforms were used: SOLiD for whole
exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the
Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH
gene regions displaying an overall coverage higher than 25×, whereas whole exome
sequencing yielded a similar coverage for only 50% of those regions. Overall
this integrated analysis led to the identification of 11 novel sequence
variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected.
However, at least two cases were not genetically solved. Our result highlights
the current limitations in the diagnostic use of NGS for USH patients. The limit
for whole exome sequencing is linked to the need of a strong coverage and to the
correct interpretation of sequence variations with a non obvious, pathogenic
role, whereas the targeted approach suffers from the high genetic heterogeneity
of USH that may be also caused by the presence of additional causative genes yet
to be identified. BACKGROUND: Gastric adenocarcinoma is a rare diagnosis in childhood. A
14-year-old male patient presented with metastatic gastric adenocarcinoma, and a
strong family history of colon cancer. Clinical sequencing of CDH1 and APC were
negative. Whole exome sequencing was therefore applied to capture the majority
of protein-coding regions for the identification of single-nucleotide variants,
small insertion/deletions, and copy number abnormalities in the patient's
germline as well as primary tumor.
MATERIALS AND METHODS: DNA was extracted from the patient's blood, primary
tumor, and the unaffected mother's blood. DNA libraries were constructed and
sequenced on Illumina HiSeq2000. Data were post-processed using Picard and
Samtools, then analyzed with the Genome Analysis Toolkit. Variants were
annotated using an in-house Ensembl-based program. Copy number was assessed
using ExomeCNV.
RESULTS: Each sample was sequenced to a mean depth of coverage of greater than
120×. A rare non-synonymous coding single-nucleotide variant (SNV) in TP53 was
identified in the germline. There were 10 somatic cancer protein-damaging
variants that were not observed in the unaffected mother genome. ExomeCNV
comparing tumor to the patient's germline, identified abnormal copy number,
spanning 6,946 genes.
CONCLUSION: We present an unusual case of Li-Fraumeni detected by whole exome
sequencing. There were also likely driver somatic mutations in the gastric
adenocarcinoma. These results highlight the need for more thorough and broad
scale germline and cancer analyses to accurately inform patients of inherited
risk to cancer and to identify somatic mutations. Discovering causative genetic variants in individual cases of suspected
mitochondrial disease requires interrogation of both the mitochondrial (mtDNA)
and nuclear genomes. Whole-exome sequencing can support simultaneous dual-genome
analysis, although currently available capture kits do not target the mtDNA
genome and provide insufficient capture for some nuclear-encoded mitochondrial
genes. To optimize interrogation of nuclear and mtDNA genes relevant to
mitochondrial biology and disease, a custom SureSelect "Mito-Plus" whole-exome
library was formulated by blending RNA "baits" from three separate designs: (A)
Agilent Technologies SureSelectXT 50 Mb All Exon PLUS Targeted Enrichment Kit,
(B) 16-gene nuclear panel targeting sequences for known MitoCarta proteins not
included in the 50 Mb All Exon design, and (C) sequences targeting the entire
mtDNA genome. The final custom formulations consisted of a 1:1 ratio of nuclear
baits to which a 1 to 1,000-fold diluted ratio of mtDNA genome baits were
blended. Patient sample capture libraries were paired-end sequenced on an
Illumina HiSeq 2000 system using v3.0 SBS chemistry. mtDNA genome coverage
varied depending on the mtDNA:nuclear blend ratio, where a 1:100 ratio provided
optimal dual-genome coverage with 10X coverage for over 97.5% of all targeted
nuclear regions and 1,000X coverage for 99.8% of the mtDNA genome. mtDNA
mutations were reliably detected to at least an 8% heteroplasmy level, as
discriminated both from sequencing errors and potential contamination from
nuclear mtDNA transcripts (Numts). The "1:100 Mito-Plus Whole-Exome" Agilent
capture kit offers an optimized tool for whole-exome analysis of nuclear and
mtDNA genes relevant to the diagnostic evaluation of mitochondrial disease. BACKGROUND: Corneal intraepithelial dyskeratosis is an extremely rare condition.
The classical form, affecting Native American Haliwa-Saponi tribe members, is
called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present
a new form of corneal intraepithelial dyskeratosis for which we identified the
causative gene by using deep sequencing technology.
METHODS AND RESULTS: A seven member Caucasian French family with two corneal
intraepithelial dyskeratosis affected individuals (6-year-old proband and his
mother) was ascertained. The proband presented with bilateral complete corneal
opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal
dyskeratosis were associated with the phenotype. Histopathology studies of
cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative
PCR ruled out 4q35 duplication, classically described in HBID cases. Next
generation sequencing with mean coverage of 50× using the Illumina Hi Seq and
whole exome capture processing was performed. Sequence reads were aligned, and
screened for single nucleotide variants and insertion/deletion calls. In-house
pipeline filtering analyses and comparisons with available databases were
performed. A novel missense mutation M77T was discovered for the gene NLRP1
which maps to chromosome 17p13.2. This was a de novo mutation in the proband's
mother, following segregation in the family, and not found in 738 control DNA
samples. NLRP1 expression was determined in adult corneal epithelium. The amino
acid change was found to destabilise significantly the protein structure.
CONCLUSIONS: We describe a new corneal intraepithelial dyskeratosis and how we
identified its causative gene. The NLRP1 gene product is implicated in
inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1
polymorphisms are associated with various diseases. Recent advances in sequencing technologies have revolutionized genetic studies.
Although high-coverage sequencing can uncover most variants present in the
sequenced sample, low-coverage sequencing is appealing for its cost
effectiveness. Here, we present AbCD (arbitrary coverage design) to aid the
design of sequencing-based studies. AbCD is a user-friendly interface providing
pre-estimated effective sample sizes, specific to each minor allele frequency
category, for designs with arbitrary coverage (0.5-30×) and sample size (20-10
000), and for four major ethnic groups (Europeans, Africans, Asians and African
Americans). In addition, we also present two software tools: ShotGun and
DesignPlanner, which were used to generate the estimates behind AbCD. ShotGun is
a flexible short-read simulator for arbitrary user-specified read length and
average depth, allowing cycle-specific sequencing error rates and realistic read
depth distributions. DesignPlanner is a full pipeline that uses ShotGun to
generate sequence data and performs initial SNP discovery, uses our previously
presented linkage disequilibrium-aware method to call genotypes, and, finally,
provides minor allele frequency-specific effective sample sizes. ShotGun plus
DesignPlanner can accommodate effective sample size estimate for any combination
of high-depth and low-depth data (for example, whole-genome low-depth plus
exonic high-depth) or combination of sequence and genotype data [for example,
whole-exome sequencing plus genotyping from existing Genomewide Association
Study (GWAS)]. PURPOSE: The management of epilepsy in children is particularly challenging when
seizures are resistant to antiepileptic medications, or undergo many changes in
seizure type over time, or have comorbid cognitive, behavioral, or motor
deficits. Despite efforts to classify such epilepsies based on clinical and
electroencephalographic criteria, many children never receive a definitive
etiologic diagnosis. Whole exome sequencing (WES) is proving to be a highly
effective method for identifying de novo variants that cause neurologic
disorders, especially those associated with abnormal brain development. Herein
we explore the utility of WES for identifying candidate causal de novo variants
in a cohort of children with heterogeneous sporadic epilepsies without etiologic
diagnoses.
METHODS: We performed WES (mean coverage approximately 40×) on 10 trios
comprised of unaffected parents and a child with sporadic epilepsy characterized
by difficult-to-control seizures and some combination of developmental delay,
epileptic encephalopathy, autistic features, cognitive impairment, or motor
deficits. Sequence processing and variant calling were performed using standard
bioinformatics tools. A custom filtering system was used to prioritize de novo
variants of possible functional significance for validation by Sanger
sequencing.
KEY FINDINGS: In 9 of 10 probands, we identified one or more de novo variants
predicted to alter protein function, for a total of 15. Four probands had de
novo mutations in genes previously shown to harbor heterozygous mutations in
patients with severe, early onset epilepsies (two in SCN1A, and one each in
CDKL5 and EEF1A2). In three children, the de novo variants were in genes with
functional roles that are plausibly relevant to epilepsy (KCNH5, CLCN4, and
ARHGEF15). The variant in KCNH5 alters one of the highly conserved arginine
residues of the voltage sensor of the encoded voltage-gated potassium channel.
In vitro analyses using cell-based assays revealed that the CLCN4 mutation
greatly impaired ion transport by the ClC-4 2Cl(-) /H(+) -exchanger and that the
mutation in ARHGEF15 reduced GEF exchange activity of the gene product,
Ephexin5, by about 50%. Of interest, these seven probands all presented with
seizures within the first 6 months of life, and six of these have intractable
seizures.
SIGNIFICANCE: The finding that 7 of 10 children carried de novo mutations in
genes of known or plausible clinical significance to neuronal excitability
suggests that WES will be of use for the molecular genetic diagnosis of sporadic
epilepsies in children, especially when seizures are of early onset and
difficult to control. Genetic alterations in specific driver genes lead to disruption of cellular
pathways and are critical events in the instigation and progression of
hepatocellular carcinoma (HCC). As a prerequisite for individualized cancer
treatment, we sought to characterize the landscape of recurrent somatic
mutations in HCC. We performed whole-exome sequencing on 87 HCCs and matched
normal adjacent tissues to an average coverage of 59×. The overall mutation rate
was roughly two mutations per Mb, with a median of 45 nonsynonymous mutations
that altered the amino acid sequence (range, 2-381). We found recurrent
mutations in several genes with high transcript levels: TP53 (18%); CTNNB1
(10%); KEAP1 (8%); C16orf62 (8%); MLL4 (7%); and RAC2 (5%). Significantly
affected gene families include the nucleotide-binding domain and leucine-rich
repeat-containing family, calcium channel subunits, and histone
methyltransferases. In particular, the MLL family of methyltransferases for
histone H3 lysine 4 were mutated in 20% of tumors.
CONCLUSION: The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in
multiple cohorts of HCC. Recent studies have demonstrated the power of deep re-sequencing of the whole
genome or exome in understanding cancer genomes. However, targeted capture of
selected genomic whole gene-body regions, rather than the whole exome, have
several advantages: 1) the genes can be selected based on biology or a
hypothesis; 2) mutations in promoter and intronic regions, which have important
regulatory roles, can be investigated; and 3) less expensive than whole genome
or whole exome sequencing. Therefore, we designed custom high-density
oligonucleotide microarrays (NimbleGen Inc.) to capture approximately 1.7 Mb
target regions comprising the genomic regions of 28 genes related to colorectal
cancer including genes belonging to the WNT signaling pathway, as well as
important transcription factors or colon-specific genes that are over expressed
in colorectal cancer (CRC). The 1.7 Mb targeted regions were sequenced with a
coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342
sequence variations in the CRC and corresponding adjacent normal tissues. Among
them, 738 were novel sequence variations based on comparisons with the SNP
database (dbSNP135). We validated 56 of 66 SNPs in a separate cohort of 30 CRC
tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of
at least 85% (56/66). We found 15 missense mutations among the exonic
variations, 21 synonymous SNPs that were predicted to change the exonic splicing
motifs, 31 UTR SNPs that were predicted to occur at the transcription factor
binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in
conserved transcription factor binding sites and 32 in CpG islands. Finally, we
determined that rs3106189, localized to the 5' UTR of antigen presenting tapasin
binding protein (TAPBP), and rs1052918, localized to the 3' UTR of transcription
factor 3 (TCF3), were associated with overall survival of CRC patients. BACKGROUND: Abdominal aortic aneurysm (AAA) is a multi-factorial disease and its
underlying pathogenesis remains poorly understood.
AIM: We aim to search for the underlying etiology of AAA using whole exome
sequencing and gene expression analysis.
MATERIALS AND METHODS: We performed whole exome sequencing for AAA and adjacent
normal abdominal aorta tissue from one male AAA patient. Further gene expression
analysis using downloaded dataset from the GEO database was also carried out to
explore the underlying molecular mechanisms.
RESULTS: A total of 5.97 Gb clean data were generated for the two samples,
achieving a mean depth of coverage of 31.96 and 32.88 for the AAA and normal
samples, respectively. We identified 203 somatic variants and confirmed 34
protein-altering somatic mutations in 25 genes. Among the confirmed variants, 11
mutations were not reported in the dbSNP database before. According to the
literature review, none of these 25 genes were reportedly associated with AAA.
CONCLUSIONS: Our findings here may provide potential targets for effective
prevention of human AAA development and progression. It has been hypothesized that, in aggregate, rare variants in coding regions of
genes explain a substantial fraction of the heritability of common diseases. We
sequenced the exomes of 1,000 Danish cases with common forms of type 2 diabetes
(including body mass index > 27.5 kg/m(2) and hypertension) and 1,000 healthy
controls to an average depth of 56×. Our simulations suggest that our study had
the statistical power to detect at least one causal gene (a gene containing
causal mutations) if the heritability of these common diseases was explained by
rare variants in the coding regions of a limited number of genes. We applied a
series of gene-based tests to detect such susceptibility genes. However, no gene
showed a significant association with disease risk after we corrected for the
number of genes analyzed. Thus, we could reject a model for the genetic
architecture of type 2 diabetes where rare nonsynonymous variants clustered in a
modest number of genes (fewer than 20) are responsible for the majority of
disease risk. |
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