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Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.	What are the advantages of the top down mass spectrometric analysis of histones?	Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain
The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor. The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparallel beta-pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the sevent invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue.	Is erabutoxin b usually found in plants?	The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map.
There have been several success stories in the field of pharmacogenetics in recent years, including the analysis of HER2 amplification for trastuzumab selection in breast cancer and VKORC1 genotyping for warfarin dosing in thrombosis. Encouraging results from these studies suggest that genetic factors may indeed be important determinants of drug response and toxicity for at least some drugs. However, to apply pharmacogenetics appropriately, a thorough understanding of the scope and limitations of this field is required. The challenges include an appreciation of biological variability, logistical issues pertaining to the proper management of information, the need for robust methods and adequate sample quality with well-designed workflows. At the same time, the economics of pharmacogenetic testing from the perspective of clinicians, patients, governments, insurance companies and pharmaceutical companies will play an important role in determining its future use. Ethical considerations such as informed consent and patient privacy, as well as the role of regulatory bodies in addressing these issues, must be fully understood. Only once these issues are properly dealt with can the full benefits of pharmacogenetics begin to be realised.	Is there a pharmacogenetic test for trastuzumab?	There have been several success stories in the field of pharmacogenetics in recent years, including the analysis of HER2 amplification for trastuzumab selection in breast cancer
Lipids are a fundamental class of organic molecules implicated in a wide range of biological processes related to their structural diversity, and based on this can be broadly classified into five categories; fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids and sphingolipids. Different lipid classes play major roles in neuronal cell populations; they can be used as energy substrates, act as building blocks for cellular structural machinery, serve as bioactive molecules, or a combination of each. In amyotrophic lateral sclerosis (ALS), dysfunctions in lipid metabolism and function have been identified as potential drivers of pathogenesis. In particular, aberrant lipid metabolism is proposed to underlie denervation of neuromuscular junctions, mitochondrial dysfunction, excitotoxicity, impaired neuronal transport, cytoskeletal defects, inflammation and reduced neurotransmitter release. Here we review current knowledge of the roles of lipid metabolism and function in the CNS and discuss how modulating these pathways may offer novel therapeutic options for treating ALS.	What links lipid metabolism pathways to ALS?	phic lateral sclerosis (ALS), dysfunctions in lipid metabolism and function have been identified as potential drivers of pathogenesis. In particu
Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.	How may CTCF mediate splicing?	Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion.
We describe an improved method for detecting deficiency of the acid hydrolase, alpha-1,4-glucosidase in leukocytes, the enzyme defect in glycogen storage disease Type II (Pompe disease). The procedure requires smaller volumes of blood and less time than previous methods. The assay involves the separation of leukocytes by Peter's method for beta-glucosidase and a modification of Salafsky and Nadler's fluorometric method for alpha-glucosidase.	Is acid alpha-glucosidase the enzyme that causes Pompe disease when mutant?	We describe an improved method for detecting deficiency of the acid hydrolase, alpha-1,4-glucosidase in leukocytes, the enzyme defect in glycogen storage disease Type II (Pompe disease).
To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.	List available genetic multicolor cell labeling techiniques in Drosophila	To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.
Etizolam is a drug from the thienotriazoldiazepine class, widely prescribed as anxiolytic due to its apparently secure toxicological profile. Nevertheless, some recent cases of etizolam dependence, intoxications and fatalities associated to its abuse have been reported in the international literature. For this reason, the drug listed as new psychoactive substance (NPS) by the World Health Organization (WHO) since 2015. Euphoric effect at high dosage is the first cause of its recreational use that has determined a wider distribution in the illicit market. An experimental study was performed to obtain evidence that etizolam at low therapeutic dosages is a drug with negligible influence on the psychomotor performances involved in driving. The psychomotor performance was assessed by performing different tests, such as critical tracking task (CTT), critical flicker fusion (CFF), choice reaction time (CRT), visual vigilance task (VVT), response competition test (RCT) in a group of 16 healthy volunteers after a single administration of etizolam at two different dosages (0.25 mg or 1.00 mg) in comparison to placebo. The test results showed that etizolam at 0.25 mg and 1.00 mg had no significant effect on vigilance, short term memory, psychomotor coordination or speed in decision making. Differently, abuse of etizolam to obtain the euphoric effects at presumably high dosages or in combination with other psychoactive substances could be fatal. The negligible side effects on mental and behavioral function demonstrated by this study, could represent an incitement to abuse, which can be strongly discouraged with correct information on differences between its correct use and its misuse.	What is Etizolam?	Etizolam is a drug from the thienotriazoldiazepine class, widely prescribed as anxiolytic due to its apparently secure toxicological profile.
Desvenlafaxine succinate (DVS) is one of several serotonin-norepinephrine reuptake inhibitors (SNRIs). Others are venlafaxine hydrochloride, milnacipran, and duloxetine. Desvenlafaxine has been approved by the US Food and Drug Administration (FDA) for the treatment of major depressive disorder (MDD) based on a number of randomized, placebo-controlled clinical trials. Clinical studies have investigated the efficacy of DVS in doses ranging from 50 to 400 mg/day for the treatment of MDD in adult outpatients. The effects of DVS 50 mg/day have been clearly distinguished from placebo in the reduction of MDD symptoms in such clinical trials. No additional therapeutic benefits were found at doses > 50 mg/day. The recommended dose of DVS ranges from 50 to 100 mg. Desvenlafaxine is currently the third SNRI approved by the FDA for this indication. Preliminary evidence also suggests the clinical usefulness of DVS in the treatment of vasomotor symptoms of menopause, anxiety symptoms, and painful physical symptoms. The modified pharmacokinetic and pharmacodynamic profiles of DVS differentiate this drug from the original product, venlafaxine. Significant points of difference, compared with venlafaxine, are once-daily dosing and the achievement of steady-state plasma concentrations within 4 to 5 days. To summarize, current evidence indicates that DVS has proven efficacy, acceptable safety and tolerability profiles, convenient dosing, and minimal impact on the cytochrome P450 enzyme system. A reduced risk for pharmacokinetic drug interactions is a potential advantage over other selective serotonin noradrenaline reuptake inhibitors. Desvenlafaxine succinate has demonstrated its efficacy for treating MDD but its variable efficacy, as shown in individual studies, limited long-term data, and its different risk-to-benefit ratio compared with earlier antidepressants, means that further investigation of this drug is necessary.	Can desvenlafaxine be used at a dose of 50mg/day?	Clinical studies have investigated the efficacy of DVS in doses ranging from 50 to 400 mg/day for the treatment of MDD in adult outpatients. The effects of DVS 50 mg/day have been clearly distinguished from placebo in the reduction of MDD symptoms in such clinical trials. No additional therapeutic benefits were found at doses > 50 mg/day. The recommended dose of DVS ranges from 50 to 100 mg.
Thirty years ago, antibodies against the muscle acetylcholine receptor (AChR) were recognized as the cause of myasthenia gravis. Since then, there has been great interest in identifying other neurological disorders associated with autoantibodies. Several other antibody-mediated neuromuscular disorders have been identified, each associated with an antibody against a ligand- or voltage-gated ion channel. The Lambert-Eaton syndrome is caused by antibodies against voltage-gated calcium channels and often occurs in patients with small cell lung cancer. Acquired neuromyotonia is caused by voltage-gated potassium channel antibodies, and autoimmune autonomic ganglionopathy is caused by antibodies against the neuronal AChR in autonomic ganglia. There is good evidence that antibodies in these disorders cause changes in synaptic function or neuronal excitability by directly inhibiting ion channel function. More recently, studies have identified ion channel antibodies in patients with certain CNS disorders, such as steroid-responsive encephalitis and paraneoplastic cerebellar ataxia. It remains unclear if antibodies can gain access to the CNS and directly cause ion channel dysfunction. Treatment of autoimmune channelopathies includes drugs that help restore normal neuronal function and treatments to remove pathogenic antibodies (plasma exchange) or modulate the immune response (steroids or immunosuppressants). These disabling neurological disorders may be dramatically responsive to immunomodulatory therapy. Future studies will likely lead to identification of other ion channel antibodies and other autoimmune channelopathies.	Which type of lung cancer is the most strongly associated with Lambert-Eaton syndrome?	The Lambert-Eaton syndrome is caused by antibodies against voltage-gated calcium channels and often occurs in patients with small cell lung cancer.
Aortitis includes conditions with infectious or non-infectious etiology, characterized by inflammatory changes in one or more layers in aortic wall. Age at onset, geographic predilections, distribution and pattern of involvement in aorta, its branches and pulmonary arteries, and systemic associations provide a clue to etiology. Clinical presentations are often non-specific. An integrated approach including clinical, laboratory and imaging assessment is essential to confirm diagnosis and plan treatment. Assessment of disease activity is the key as it influences timing and outcome of treatment. Markers of activity include clinical, laboratory and imaging. Medical management remains the first-line therapy. Revascularization is indicated in the presence of hemodynamically significant stenosis and inactive disease. In the presence of flash pulmonary edema, left ventricular dysfunction or hypertensive encephalopathy, revascularization is performed irrespective of disease activity. Endovascular management is favored over surgery due to its high success and low restenosis rates. Symptomatic aneurysmal disease is usually managed by surgery.	What is Aortitis?	Aortitis includes conditions with infectious or non-infectious etiology, characterized by inflammatory changes in one or more layers in aortic wall.
The LINCS L1000 data repository contains almost two million gene expression profiles for thousands of small molecules and drugs. However, due to the complexity and the size of the data repository and a lack of an interoperable interface, the creation of pharmacologically meaningful workflows utilizing these data is severely hampered. In order to overcome this limitation, we developed the L1000 Viewer, a search engine and graphical web interface for the LINCS data repository. The web interface serves as an interactive platform allowing the user to select different forms of perturbation profiles, e.g., for specific cell lines, drugs, dosages, time points and combinations thereof. At its core, our method has a database we created from inferring and utilizing the intricate dependency graph structure among the data files. The L1000 Viewer is accessible via http://L1000viewer.bio-complexity.com/.	The LINCS L1000 data set contains gene expression data for drug treated human cells, yes or no?	The LINCS L1000 data repository contains almost two million gene expression profiles for thousands of small molecules and drugs.
Malignant melanomas in black Africans are predominantly located on the lower extremities. Since their biological features have not been well focused, we studied 28 such cases with special reference to proliferative activity (Ki-67 expression), p16 and p53 staining, as well as microvessel density, all known to be involved in the progression of melanomas among whites. The findings were related to clinico-pathological characteristics. The tumours had a median thickness of 6.4 mm, ulceration was present in 71%, and vascular invasion in 36%, indicating the presence of advanced and aggressive melanomas. Further, loss of p16 protein expression was found in 50%, and high proliferative activity was present (median 41%). In contrast, strong p53 staining was rare (11%), although most tumours showed low-level positivity. Angiogenesis, as estimated by microvessel density, was significantly associated with vascular invasion (p = 0.022), supporting its role in the progression of these tumours. Thus, our findings indicate that melanomas located on the lower extremities in black Africans show several features of aggressiveness; in particular, the proliferative activity was high, and p16 alterations was frequent as evidenced by loss of protein staining. Our findings also indicated that the diagnosis is delayed among black Africans.	Does melanoma occur in people of African origin ?	Malignant melanomas in black Africans are predominantly located on the lower extremities
Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.	What is the ChIP-exo method used for?	The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site.
The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer-dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.	Which proteins does the p85α interact with?	p110α-free p85α homodimerizes
This pilot study indicates that telemedicine follow-up consultations for chronic pain patients are feasible and cost-saving. Patients and anesthesiologists were highly satisfied with telemedicine consultation. Patients reported a significant saving in time and cost compared with a conventional consultation.	Are there telemedicine applications for chronic pain management?	This pilot study indicates that telemedicine follow-up consultations for chronic pain patients are feasible and cost-saving. Patients and anesthesiologists were highly satisfied with telemedicine consultation.
Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor with a median survival of 1 year after diagnosis. It has been reported recently that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed. Here, we describe a real-time PCR-based procedure involving a mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can differentiate samples with H3F3A K27M mutation from those that do not. We first tested four different mutant-specific primers for their ability to selectively amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant allele more efficiently than the rest. We then determined the optimal concentration of blocker oligo that significantly improved amplification of the H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed eleven samples, two of which containing H3F3A K27M mutation, and found that these two samples were differentially amplified from the nine others. In addition, we were able to discern the H3F3A K27M mutation in a newly obtained pediatric brainstem glioblastoma sample whose H3.3 status was not known previously, and in three other DIPG samples as well as paraffin embedded samples. These results demonstrate that we have developed a new reliable procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma patient samples.	Which histone mutations have been associated with pediatric gliomas?	In order to facilitate diagnosis of DIPG patients, a quick and reliable method to identify the H3F3A K27M mutation is needed.
Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.	Which enzymes are responsible for base J creation in Trypanosoma brucei?	JBP2 and JBP1 are capable of stimulating de novo J-synthesis.
In this study, we took advantage of the overexpression of human epidermal growth factor receptor 2 (HER-2) in prostate cancers to design lentiviruses with modified envelope proteins that bind antibodies to specific cell-surface antigens. When bound to trastuzumab (Herceptin, Genentech, CA), lentiviruses were able to selectively infect androgen-sensitive LNCaP and castration-resistant C4-2 human prostate cancer cell lines, both of which express high levels of HER-2. To test for a therapeutic effect, we engineered our antibody-binding lentiviruses to express thymidine kinase, which can convert the non-toxic pro-drug ganciclovir (GCV) into a cytotoxic form. LNCaP and C4-2 cells infected by these viruses were sensitive to GCV killing. In vivo, C4-2 xenograft tumors treated either intratumorally or i.v. with trastuzumab-bound lentivirus expressed luciferase, although the latter route was less tumor specific. When a prostate-specific promoter for governing luciferase expression was combined with trastuzumab-mediated delivery, there was a further enrichment in targeting viral gene expression in prostate tumors. In conclusion, we found that although prostate cancers that express high levels of HER-2 are resistant to the killing effects of trastuzumab, they can be targeted for selective gene expression and destruction by viruses with envelope proteins engineered to bind this antibody.	Is the monoclonal antibody Trastuzumab (Herceptin) of potential use in the treatment of prostate cancer?	we found that although prostate cancers that express high levels of HER-2 are resistant to the killing effects of trastuzumab, they can be targeted for selective gene expression and destruction by viruses with envelope proteins engineered to bind this antibody
Necroptosis is an inflammatory form of programmed cell death mediated by the pseudokinase mixed-lineage kinase domain-like protein (MLKL). Upon phosphorylation by receptor-interacting protein kinase-3 (RIPK3), MLKL oligomerizes, and translocates to and disrupts the plasma membrane, thereby causing necroptotic cell lysis. Herein, we show that activation of necroptosis in mouse dermal fibroblasts (MDFs) and HT-29 human colorectal cancer cells results in accumulation of the autophagic marker, lipidated LC3B (also known as MAP1LC3B), in an MLKL-dependent manner. Unexpectedly, the necroptosis-induced increase in lipidated LC3B was due to inhibition of autophagic flux, not the activation of autophagy. Inhibition of autophagy by MLKL correlated with a decrease in autophagosome and/or autolysosome function, and required the association of activated MLKL with intracellular membranes. Collectively, our findings uncover an additional role for the MLKL pseudokinase, namely to inhibit autophagy during necroptosis.	Do cells undergoing necroptosis show disruption of their cell membranes?	anslocates to and disrupts the plasma membrane, thereby causing necroptotic cell lysis. Herein, we show that activation of necroptosis in mouse dermal
Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis, not only affect the intestinal tract but also have an extraintestinal involvement within the oral cavity. These oral manifestations may assist in the diagnosis and the monitoring of disease activity, whilst ignoring them may lead to an inaccurate diagnosis and useless and expensive workups. Indurated tag-like lesions, cobblestoning, and mucogingivitis are the most common specific oral findings encountered in CD cases. Aphthous stomatitis and pyostomatitis vegetans are among non-specific oral manifestations of IBD. In differential diagnosis, side effects of drugs, infections, nutritional deficiencies, and other inflammatory conditions should also be considered. Treatment usually involves managing the underlying intestinal disease. In severe cases with local symptoms, topical and/or systemic steroids and immunosuppressive drugs might be used.	Is there an association between pyostomatitis vegetans and Crohn's disease?	Aphthous stomatitis and pyostomatitis vegetans are among non-specific oral manifestations of IBD.
Regeneration of articular cartilage is of great interest in cartilage tissue engineering since articular cartilage has a low regenerative capacity. Due to the difficulty in obtaining healthy cartilage for transplantation, there is a need to develop an alternative and effective regeneration therapy to treat degenerative or damaged joint diseases. Stem cells including various adult stem cells and pluripotent stem cells are now actively used in tissue engineering. Here, we provide an overview of the current status of cord blood cells and induced pluripotent stem cells derived from these cells in cartilage regeneration. The abilities of these cells to undergo chondrogenic differentiation are also described. Finally, the technical challenges of articular cartilage regeneration and future directions are discussed.	What cells are used to regenerate cartilage?	Here, we provide an overview of the current status of cord blood cells and induced pluripotent stem cells derived from these cells in cartilage regeneration.
Ocular toxoplasmosis is present in 20% of infected immunocompetent individuals. Toxoplasmosis is the most common cause of posterior uveitis in immunocompetent subjects and congenital toxoplasmosis transmission was the first parasite to be linked to human lesions in the eye. An experimental model for congenital ocular toxoplasmosis was developed in C57BL/6 mice with the purpose to evaluate Toxoplasma induced ocular pathology during fetal life. Toxoplasma gondii, ME-49 strain, was used to infect pregnant females. Histological analysis of pre-natal fetal eyes from infected female mice, did not show parasite infestation, however, alterations were observed in the outer nuclear layer (ONL) and in the inner nuclear layers (INL) of the retina. Edema was also observed, characterized by the increase of interstitial spaces forming lacunae between the ONL and INL cells and a net of vessels associated with an intense inflammatory infiltrate. These histological observations suggest that ocular lesions are not delayed manifestations of toxoplasmosis. The eye was affected in the initial phase of disease, and these alterations were of similar nature as those observed in mice at later stages of infection.	What eye disease(s) are associated with ocular toxoplasmosis?	Toxoplasmosis is the most common cause of posterior uveitis in immunocompetent subjects and congenital toxoplasmosis transmission was the first parasite to be linked to human lesions in the eye.
Human psoriasin (S100A7) has originally been described as a member of the family of S100 calcium-binding proteins which is overexpressed in patients suffering from psoriasis. The bovine homolog was first identified as a cow-derived respiratory allergen. As Escherichia coli mastitis is a common problem in dairy cattle, and human psoriasin was found to exhibit antimicrobial activity preferentially against E. coli, we examined whether the bovine mRNA is expressed in the mammary gland. To demonstrate the antimicrobial activity of bovine psoriasin, we isolated cDNA from the udder, cloned the bovine psoriasin gene in a bacterial expression vector, and the recombinant protein was expressed in BL21 cells. The in vitro antibacterial activity was tested by performing microdilution susceptibility tests and radial diffusion assays with eight different bacterial strains, thereof three different E. coli strains, and one yeast. The antimicrobial activity of the recombinant bovine psoriasin is comparable with human psoriasin and also limited to E. coli. Psoriasin appears to be a part of the local host defense mechanism in the udder, is a putative candidate for a cow-specific factor influencing mastitis susceptibility, and a possible alternative to conventional antibiotics.	In which condition was protein S100A7 originally identified?	Human psoriasin (S100A7) has originally been described as a member of the family of S100 calcium-binding proteins which is overexpressed in patients suffering from psoriasis.
Giant cell tumor of bone (GCTB) is a rare primary bone tumor that primarily affects young adults, but can be seen in children. The primary modality of treatment is surgical resection; however, this is not always possible given the location and extent of the neoplasm. Recent developments in the understanding of the underlying molecular pathogenesis of disease have pointed to interactions between the stromal component producing receptor activator of nuclear factor-kappaB (RANK) and RANK-ligand (RANKL) causing the formation of osteoclast-like giant cells that drive bone destruction. The development of a monoclonal humanized antibody to RANKL, denosumab, has been shown to reduce skeletal-related events from osteoporosis and from bony metastases from solid tumors. Recent phase II clinical trials with denosumab in skeletally mature adolescents over age 12 years and adults with GCTB, have shown both safety and efficacy, leading to its accelerated US FDA approval on 13 June 2013. In children who are skeletally immature, safety and efficacy has not been established, and there has been only published anecdotal use.	Has Denosumab (Prolia) been approved by FDA?	Recent phase II clinical trials with denosumab in skeletally mature adolescents over age 12 years and adults with GCTB, have shown both safety and efficacy, leading to its accelerated US FDA approval on 13 June 2013.
Amyotrophic lateral sclerosis (ALS) is found in a familial form in around 5-10% of cases. Of these familial cases around 20% are associated with mutations of SOD-1. The genetic basis of the disease in the remaining familial cases, and genetic risk factors in sporadic cases, are unknown. Recently, the common forms of spinal muscular atrophy (SMA) have been associated with mutations of the SMN and NAIP genes on chromosome 5, in the region q11.2-13.3. Some patients with both familial and sporadic motor neuron disease show only lower motor neuron signs, in common with SMA patients, and families containing individuals with phenotypes of both childhood SMA and adult motor neuron disease have been reported. We therefore examined the SMA locus as a candidate for ALS, in 54 patients with sporadic motor neuron disease, and 10 single-generation familial patients (with no evidence of SOD-1 mutations), and in a single patient with Brown-Vialetto-Van Laere syndrome. No mutations of the SMN or NAIP genes were detected. The difficulties of classification of lower motor neuron presentations of motor neuron diseases are discussed. The demonstration that mutations diagnostic of SMA are not found in ALS patients helps distinguish these conditions.	Which is the genetic basis of Spinal Muscular Atrophy (SMA)?	Recently, the common forms of spinal muscular atrophy (SMA) have been associated with mutations of the SMN and NAIP genes on chromosome 5, in the region q11.2-13.3.
In mammalian oocytes, cyclic AMP-dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIalpha in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIalpha protein was reduced to non-detectable levels, compensatory decreases were also observed in the RIIalpha and catalytic (Calpha) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Calpha was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis.	What are the main results of PRKAR1A Knockdown?	These results demonstrate that RIalpha is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur.
Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4-11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.	Which molecules are inhibited by Gilteritinib?	These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.
The anti-apoptotic transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in CD34(+) myeloblasts from high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Inhibition of NF-kappaB by suppressing the canonical NF-kappaB activation pathway, for instance by knockdown of the three subunits of the inhibitor of NF-kappaB (I kappaB) kinase (IKK) complex (IKK1, IKK2 and NEMO) triggers apoptosis in such cells. Here, we show that an MDS/AML model cell line exhibits a constitutive interaction, within the nucleus, of activated, S1981-phosphorylated ataxia telangiectasia mutated (ATM) with NEMO. Inhibition of ATM with two distinct pharmacological inhibitors suppressed the activating autophosphorylation of ATM, blocked the interaction of ATM and NEMO, delocalized NEMO as well as another putative NF-kappaB activator, PIDD, from the nucleus, abolished the activating phosphorylation of the catalytic proteins of the IKK complex (IKK1/2 on serines 176/180), enhanced the expression of I kappaB alpha and caused the relocalization of NF-kappaB from the nucleus to the cytoplasm, followed by apoptosis. Knockdown of ATM with small-interfering RNAs had a similar effect that could not be enhanced by knockdown of NEMO, PIDD and the p65 NF-kappaB subunit, suggesting that an ATM inhibition/depletion truly induced apoptosis through inhibition of the NF-kappaB system. Pharmacological inhibition of ATM also induced the nucleocytoplasmic relocalization of p65 in malignant myeloblasts purified from patients with high-risk MDS or AML, correlating with the induction of apoptosis. Altogether, these results support the contention that constitutively active ATM accounts for the activation of NF-kappaB in high-risk MDS and AML.	Which are the subunits of the IkB protein kinase (IKK)?	Inhibition of NF-kappaB by suppressing the canonical NF-kappaB activation pathway, for instance by knockdown of the three subunits of the inhibitor of NF-kappaB (I kappaB) kinase (IKK) complex (IKK1, IKK2 and NEMO) triggers apoptosis in such cells.
With the recent introduction of inhibitors of mammalian target of rapamycin (mTOR) in oncology, distinct cutaneous and oral adverse events have been identified. In fact, stomatitis and rash are documented as the most frequent and potentially dose-limiting side effects. Clinically, mTOR inhibitor-associated stomatitis (mIAS) more closely resembles aphthous stomatitis than oral mucositis due to conventional anticancer therapies. While most cases of mIAS are mild to moderate and self-limiting, more severe and persistent mIAS can become a dose-limiting toxicity. Small ulcerations may cause significant pain and mucosal sensitivity may occur in the absence of clinical changes. Use of clinical assessment tools that are primarily driven by ulceration size may underestimate mIAS, and assessment should include patient-reported outcomes. This article provides an up-to-date review of the clinical presentation, terminology, pathogenesis, assessment and management of mIAS and other mTOR inhibitor-associated oral adverse events. In addition, areas of future research are considered.	What does mTOR stands for?	mammalian target of rapamycin (mTOR)
Lipid lowering drugs, such as statins, are commonly used to treat approximately 10 million Canadians affected by hypercholesterolemia. The most commonly experienced side-effect of statin medication is muscle pain. Statin induced myopathy consists of a spectrum of myopathic disorders ranging from mild myalgia to fatal rhabdomyolysis. The following is a presentation of 2 cases of statin induced myopathy in patients presenting in a chiropractic setting. In addition, discussion will surround the mechanism, predisposing risk factors and frequency of statin induced myopathy while highlighting the role that chiropractors and other manual therapists may play in its recognition and management.	What class of drugs frequently has muscle pain and other muscle toxicities such as mysositis and rhabdomyolysis as a side effect?	The following is a presentation of 2 cases of statin induced myopathy in patients
Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1(-/-) primary glia upon incubation with fatty acids was reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.	What is the lipid droplet used for in the cell?	Lipid droplets are found in all cell types
Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. Hepcidin, a negative regulator of intestinal iron absorption, is found to be inappropriately low in both patients and in animal models, indicating that proper control of basal hepcidin levels requires both hemojuvelin and HFE. In mice, repulsive guidance molecule c (Rgmc, the hemojuvelin mouse ortholog) and hepcidin levels are transcriptionally regulated during inflammation. Here, we report that basal Rgmc levels in Hfe-deficient mice are normal and that these mice retain the ability to suppress Rgmc expression after lipopolysaccharide (LPS) challenge. Thus, Rgmc regulation by LPS is Hfe-independent. The response of Rgmc to LPS involves signaling through toll-like receptor 4 (Tlr4), because Tlr4-deficient mice do not show altered Rgmc expression after LPS administration. We further show that tumor necrosis factor-alpha, but not interleukin-6, is sufficient to cause Rgmc down-regulation by LPS. These results contrast with previous data demonstrating that hepcidin levels are directly regulated by interleukin-6 but not by tumor necrosis factor-alpha. The regulation of iron-related genes by different cytokines may allow for time-dependent control of iron metabolism changes during inflammation and may be relevant to chronic inflammation, infections, and cancer settings, leading to the development of anemia of chronic disease.	Which conditions is caused by mutations in HFE?	Genetic iron overload, or hemochromatosis, can be caused by mutations in HFE, hemojuvelin, and hepcidin genes. He
The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA-protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1-DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1-DNA complexes.	What is the SPRTN protein function?	we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs.
The SWR1/SRCAP complex is a chromatin-remodeling complex that has been shown to be involved in substitution of histone H2A by the histone variant H2A.Z in yeast (Saccharomyces cerevisiae) and animals. Here, we identify and characterize SERRATED LEAVES AND EARLY FLOWERING (SEF), an Arabidopsis (Arabidopsis thaliana) homolog of the yeast SWC6 protein, a conserved subunit of the SWR1/SRCAP complex. SEF loss-of-function mutants present a pleiotropic phenotype characterized by serrated leaves, frequent absence of inflorescence internodes, bushy aspect, and flowers with altered number and size of organs. sef plants flower earlier than wild-type plants both under inductive and noninductive photoperiods. This correlates with strong reduction of FLOWERING LOCUS C and MADS-AFFECTING FLOWERING4 transcript levels and up-regulation of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene expression. The sef phenotype is similar to that of the photoperiod-independent early flowering1 (pie1) and the actin-related protein 6 (arp6) mutants. PIE1 and ARP6 proteins are also homologs of SWR1/SRCAP complex subunits. Analysis of sef pie1 double mutants demonstrates genetic interaction between these two genes. We also show physical interactions between SEF, ARP6, and PIE1 proteins. Taken together, our data indicate that SEF, ARP6, and PIE1 might form a molecular complex in Arabidopsis related to the SWR1/SRCAP complex identified in other eukaryotes.	Which protein mediates the replacement of H2A by H2A.Z in the yeast Saccharomyces cerevisiae?	Here, we identify and characterize SERRATED LEAVES AND EARLY FLOWERING (SEF), an Arabidopsis (Arabidopsis thaliana) homolog of the yeast SWC6 protein, a conserved subunit of the SWR1/SRCAP complex.
This network meta-analysis compared the short-term and long-term efficacies of first-line chemotherapy regimens in patients with advanced colorectal cancer (CRC). The 10 regimens included folinic acid + 5-fluorouracil + oxaliplatin (FOLFOX), folinic acid + 5-fluorouracil + irinotecan (FOLFIRI), folinic acid + 5-fluorouracil + gemcitabine (FFG), folinic acid + 5-fluorouracil + trimetrexate (FFT), folinic acid + 5-fluorouracil (FF), irinotecan + oxaliplatin (IROX), raltitrexed + oxaliplatin (TOMOX), folinic acid + tegafur-uracil (FTU), raltitrexed, and capecitabine. Electronic searches were performed in the Cochrane Library, PubMed and Embase databases from inception to June 2017. Network meta-analysis combined direct and indirect evidence to obtain odds ratios (ORs) and surface under the cumulative ranking curves (SUCRA) of different chemotherapy regimens for advanced CRC. Fourteen randomized controlled trails (RCTs) covering 4,383 patients with advanced CRC were included. The results revealed that FOLFOX, FOLFIRI, IROX, and TOMOX all showed higher overall response rates (ORRs) than FF or raltitrexed. Compared with raltitrexed, the aforementioned four regimens also had higher 1-year progression-free survival (PFS) rates. In addition, FOLFOX and FOLFIRI exhibited higher disease control rates (DCRs) and 1-year PFS rates than FF or raltitrexed. Cluster analysis revealed that FOLFOX, FOLFIRI, and TOMOX had better short-term and long-term efficacies. These findings suggest FOLFOX, FOLFIRI, and TOMOX are superior to other regimens for advanced CRC. These three regimens are therefore recommended for clinical treatment of advanced CRC.	Which drugs are included in the IROX regimen for colorectal cancer?	The 10 regimens included folinic acid + 5-fluorouracil + oxaliplatin (FOLFOX), folinic acid + 5-fluorouracil + irinotecan (FOLFIRI), folinic acid + 5-fluorouracil + gemcitabine (FFG), folinic acid + 5-fluorouracil + trimetrexate (FFT), folinic acid + 5-fluorouracil (FF), irinotecan + oxaliplatin (IROX), raltitrexed + oxaliplatin (TOMOX), folinic acid + tegafur-uracil (FTU), raltitrexed, and capecitabine.
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. Given the systemic nature of DS-related features, it is natural to attribute neonatal complications to the chromosomal aberration. We describe a biracial male infant with DS who had significantly delayed defecation and required continuous ventilator support, but had no primary cardiac or lung disease. Subsequent evaluations confirmed total colonic aganglionosis. Because we were unable to safely extubate the infant, a diagnosis of congenital central hypoventilation syndrome (CCHS) was considered and confirmed by molecular analysis of the PHOX2B gene, revealing a heterozygous polyalanine repeat-expansion mutation containing 27 repeats (normal gene contains 20 repeats). HSCR coexisting with CCHS is known as Haddad syndrome. This is the first reported case with co-occurrence of DS, CCHS, and HSCR.	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
Ambras syndrome (AS) is a rare form of congenital hypertrichosis with excessive hair on the shoulders, face and ears. Cytogenetic studies have previously implicated an association with rearrangements of chromosome 8. Here we define an 11.5 Mb candidate interval for AS on chromosome 8q based on cytogenetic breakpoints in three patients. TRPS1, a gene within this interval, was deleted in a patient with an 8q23 chromosomal rearrangement, while its expression was significantly downregulated in another patient with an inversion breakpoint 7.3 Mb downstream of TRPS1. Here, we describe the first potential long-range position effect on the expression of TRPS1. To gain insight into the mechanisms by which Trps1 affects the hair follicle, we performed a detailed analysis of the hair abnormalities in Koa mice, a mouse model of hypertrichosis. We found that the proximal breakpoint of the Koa inversion is located 791 kb upstream of Trps1. Quantitative real-time polymerase chain reaction, in situ hybridization and immunofluorescence analysis revealed that Trps1 expression levels are reduced in Koa mutant mice at the sites of pathology for the phenotype. We determined that the Koa inversion creates a new Sp1 binding site and translocates additional Sp1 binding sites within a highly conserved stretch spanning the proximal breakpoint, providing a potential mechanism for the position effect. Collectively, these results describe a position effect that downregulates TRPS1 expression as the probable cause of hypertrichosis in AS in humans and the Koa phenotype in mice.	Describe clinical presentation of Ambras syndrome.	Ambras syndrome (AS) is a rare form of congenital hypertrichosis with excessive hair on the shoulders, face and ears.
Telomerase is expressed in 85-90 % of pancreatic adenocarcinomas and might be a target for active cancer immunotherapy. A study was conducted to investigate safety and immunogenicity in non-resectable pancreatic carcinoma patients using a 16-amino acid telomerase peptide (GV1001) for vaccination in combination with GM-CSF and gemcitabine as first line treatment. Three different vaccine treatment schedules were used; [A (n=6), B (n=6) and C (n=5)]. Groups A/B received GV1001, GM-CSF and gemcitabine concurrently. Group C received initially GV1001 and GM-CSF while gemcitabine was added at disease progression. Group D (n=4) was treated with gemcitabine alone. Adverse events (AE) related to vaccination were mild (grades I-II). Grade III AEs were few and transient. An induced GV 1001‑specific immune response was defined as an increase ≥2 above the baseline value in one of the assays (DTH, proliferation, ELISPOT and cytokine secretion assays, respectively). A telomerase‑specific immune response was noted in 4/6 patients in group A, 4/6 patients in group B and 2/5 patients in group C. An induced ras‑specific immune response (antigenic spreading) was seen in 5 of the 17 patients. The cytokine pattern was that of a Th1-like profile. A treatment induced telomerase or ras response was also noted in group D. All responses were weak and transient. A significant decrease in regulatory T-cells over time was noted in patients in groups A and B (p<0.05). Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient. Measures have to be taken to optimize immune responses of GV1001 for it to be considered of clinical interest.	GV1001 vaccine targets which enzyme?	Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient.
Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.	What is the reason for N-acetylgalactosamine (GalNAc) conjugation of siRNAs?	Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes.