Context: Dementia with Lewy bodies (DLB) is characterized by the widespread presence of Lewy bodies (LBs) in the brain. alpha-Synuclein, the main component of LBs, is expressed as two main isoforms (112 and 140), but little is known about their differential expression in the brain. We compared alpha-synuclein 112 and alpha-synuclein 140 expression levels in the prefrontal cortices of six DLB patients, eight Alzheimer disease (AD) patients, and six control subjects. Relative alpha-synuclein 112 and alpha-synuclein 140 expression levels were determined by real-time polymerase chain reaction with competimer technology using a LightCycler System. Whereas total alpha-synuclein levels were just marginally elevated in DLB in comparison with the other groups, alpha-synuclein 112 was seen to be markedly increased in DLB compared with AD cases and controls. In contrast, alpha-synuclein 140 levels were significantly diminished in both neurodegenerative disorders in comparison with controls. These results show differential overexpression of alpha-synuclein 112 in DLB, a finding that could be of importance in DLB pathogenesis.


Context: We have demonstrated that both high-K+ and electrical stimulation of the vagus nerves release endogenous glutamate from the vascularly-perfused rat stomach in a calcium-dependent manner. In the present study, we examined properties of calcium channel subtypes mediating endogenous glutamate release from the stomach. Application of 50 mM KCl elicited a release of glutamate, and this release was abolished in calcium-free medium. The release of glutamate was significantly inhibited by both omega-agatoxin IVA, a P/Q-type calcium channel antagonist, and isradipine, an L type calcium channel antagonist. Omega-conotoxin GVIA, an N type calcium channel antagonist and flunarizine, a nonselective T-type calcium channel antagonist were without effect. In contrast to this case of glutamate, omega-conotoxin GVIA induced a marked inhibition in the release of gastric noradrenaline. The combined treatment with omega-agatoxin IVA plus isradipine produced a marked synergistic inhibition of the glutamate release. This inhibition was, however, much less than that by cadmium. The present results suggest that P/Q and L type calcium channels coexist to regulate the release of gastric glutamate. Furthermore, it is possible that unidentified calcium channels other than P/Q and L type channels are also involved in the release of glutamate in the stomach.


Context: The p15 and p16 CDK4 inhibitor genes map within the chromosome band 9p21 region deleted frequently in malignant mesothelioma and other cancers. p16 has been implicated recently as a potential target of 9p21 deletions in mesothelioma, but the role of this gene is uncertain because deletions have been detected more often in established cell lines than in primary tumor specimens. We determined p15 and p16 copy number by fluorescence in situ hybridization with a P1 contig in 50 primary mesotheliomas. Codeletion of p15 and p16 was found in 72% of mesotheliomas, including all cases with spindle-cell components (n = 21) and total deletion of p15 and p16 was found in several mesotheliomas that lacked cytogenetic deletion of the chromosome 9 short arm. Point mutations were not found, however, in exon 2 of retained p15 and p16 alleles from seven mesotheliomas. These findings demonstrate that p15, p16 and/or a closely neighboring gene, are the targets of frequent chromosome 9p deletion in primary malignant mesothelioma.


Context: The enzyme responsible for the conversion of phosphatidylglycerol to diphosphatidylglycerol (cardiolipin) in the presence of cytidine diphosphate diacylglycerol is firmly associated with mitochondrial membranes and is not extracted with hypotonic or hypertonic media or with nonionic detergents. Some solubilization was obtained with bile salt solutions, but the zwitter-ionic detergent. Miranol H2M, was most effective in extracting the enzyme. The Miranol extracts were fractionated by column chromatography on Bio-Gel A-1.5 m. The solubilized enzyme is considerably more active in converting unsaturated than saturated phosphatidyl-glycerols, but shows little preference for the cytidine diphosphate diacylglycerols with different fatty acyl substituents. There is an absolute dependence upon divalent cations with the order of effectiveness: Co2+ much greater than Mn2+ greater than Mg2+. In the presence of optimal levels of Co2+ other divalent cations are inhibitory with the order of inhibition: Cd2+ greater than Zn2+ greater than Ca2+ greater than Ba2+ greater than Cu2+ greater than Hg2+ greater than Ni2+. The solubilized enzyme exhibited no requirement for added phospholipids and several phospholipids inhibited the reaction in the order: diphosphatidylglycerol greater than phosphatidylethanolamine greater than phosphatidylserine greater than phosphatidylinositol.


Context: The overexpression of ATP-binding cassette (ABC) transporters has been proved to be a major trigger for multidrug resistance (MDR) in certain types of cancer. In our study, we investigated whether osimertinib (AZD9291), a third-generation irreversible tyrosine kinase inhibitor of both activating EGFR mutations and resistance-associated T790M point mutation, could reverse MDR induced by ABCB1 and ABCG2 in vitro, in vivo, and ex vivo Our results showed that osimertinib significantly increased the sensitivity of ABCB1- and ABCG2-overexpressing cells to their substrate chemotherapeutic agents in vitro and in the model of ABCB1-overexpressing KBv200 cell xenograft in nude mice. Mechanistically, osimertinib increased the intracellular accumulations of doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, osimertinib stimulated the ATPase activity of both ABCB1 and ABCG2 and competed with the [(125)I] iodoarylazidoprazosin photolabeling bound to ABCB1 or ABCG2, but did not alter the localization and expression of ABCB1 or ABCG2 in mRNA and protein levels nor the phosphorylations of EGFR, AKT, and ERK. Importantly, osimertinib also enhanced the cytotoxicity of DOX and intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. Overall, these findings suggest osimertinib reverses ABCB1- and ABCG2-mediated MDR via inhibiting ABCB1 and ABCG2 from pumping out chemotherapeutic agents and provide possibility for cancer combinational therapy with osimertinib in the clinic. Mol Cancer Ther; 15(8); 1845-58. ©2016 AACR.


Context: Teriparatide (recombinant human parathyroid hormone ) is an anabolic agent approved for the treatment of patients at high risk for fracture. The Fracture Prevention Trial administered teriparatide to treatment-naïve patients, leading to its US Food and Drug Administration approval in 2002. Clinical trial data using antiresorptive agents administered before, during, and after any parathyroid hormone (PTH) therapy, as well as alternative PTH dosing, have provided additional insight yet raise fundamental questions about the most appropriate use of teriparatide. This article provides an update on teriparatide, focusing on its mechanism of action compared with other antiresorptive agents, indications, adverse effects, therapy duration, combination therapy, contraindications, and cost effectiveness.


Context: Methotrexate, a folate antagonist, is a potent antiinflammatory agent when used weekly in low concentrations. We examined the hypothesis that the antiphlogistic effects of methotrexate result from its capacity to promote intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) that, under conditions of cell injury, increases local adenosine release. We now present the first evidence to establish this mechanism of action in an in vivo model of inflammation, the murine air pouch model. Mice were injected intraperitoneally with either methotrexate or saline for 3-4 wk during induction of air pouches. Pharmacologically relevant doses of methotrexate increased splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibited leukocyte accumulation in inflamed air pouches. The methotrexate-mediated reduction in leukocyte accumulation was partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reversed by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine. Neither ADA nor DMPX affected leukocyte accumulation in the inflamed pouches of animals treated with either saline or the potent antiinflammatory steroid dexamethasone. These results indicate that methotrexate is a nonsteroidal antiinflammatory agent, the antiphlogistic action of which is due to increased adenosine release at inflamed sites.


Context: Pembrolizumab (Keytruda(®)) is a humanized monoclonal antibody against programmed death receptor-1 (PD-1), a key immunoinhibitory checkpoint protein implicated in down-regulating anti-tumour immune responses. This intravenous drug is indicated for the treatment of advanced (unresectable or metastatic) melanoma, on the basis of its clinical benefit in this setting in the phase I KEYNOTE 001 trial (expansion cohorts) and the phase II and III trials, KEYNOTE 002 and 006. These studies were conducted in ipilimumab-naïve and/or ipilimumab-experienced patients and assessed varying pembrolizumab regimens administered every 2 or 3 weeks, all of which helped to determine the recommended dosage of 2 mg/kg every 3 weeks. In the trials with active comparator arms, pembrolizumab regimens significantly improved progression-free survival (PFS), overall survival (OS) and overall response rates (ORR) relative to ipilimumab in ipilimumab-naïve patients (KEYNOTE 006), and significantly improved PFS and ORR, but not OS (although OS data are immature), relative to chemotherapy in ipilimumab-refractory patients, who had also received BRAF/MEK inhibitor therapy if BRAF-mutation positive (KEYNOTE 002). Pembrolizumab has an acceptable tolerability profile, with immune-related adverse events that are generally manageable/reversible. Thus, pembrolizumab is a valuable treatment option for patients with advanced melanoma, including those who have progressed on ipilimumab and BRAF/MEK inhibitors.


Context: Ultraconserved elements (UCEs) encoding noncoding RNAs serve as important regulators in cancer biology. Until now, the role of the UCE uc.189 in human cancers remains undefined and the clinical significance of uc.189 in esophageal cancers remains unknown. This study was to identify the prognostic value of uc.189 expression in esophageal squamous cell carcinomas (ESCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of uc.189 in matched cancerous tissues and adjacent noncancerous tissues from 152 patients with ESCC. The correlation of uc.189 with clinicopathological features and prognosis were also analyzed. The expression of uc.189 was significantly higher in human ESCC compared with the adjacent noncancerous tissues (122/152, 80.3%, p<0.01), and the high level of uc.189 expression was significantly correlated with invasion of the tumor (p=0.009), advanced clinical stage (p=0.000), lymph node metastasis (p=0.000), and poor prognosis. High expression of uc.189 might reflect poor prognosis of ESCC and indicate a potential diagnostic target in ESCC patients. Uc.189 might be considered as a novel molecule involved in ESCC progression, which provides a potential prognostic biomarker and therapeutic target.


Context: The direct thrombin inhibitor dabigatran and the anti-Xa agents rivaroxaban, edoxaban, and apixaban are a new generation of oral anticoagulants. Their advantage over the vitamin K antagonists is the lack of the need for monitoring and dose adjustment. Their main disadvantage is currently the absence of a specific reversal agent. Dabigatran's, unlike the anti-Xa agents, absorption can be reduced by activated charcoal if administered shortly after ingestion and it can be removed from the blood with hemodialysis. Prothrombin complex concentrate, activated prothrombin complex concentrate, and recombinant factor VIIa all show some activity in reversing the anticoagulant effect of these drugs but this is based on ex vivo, animal, and volunteer studies. It is unclear, which, if any, of these drugs is the most suitable for emergency reversal. Three novel molecules (idarucizumab, andexanet, and PER977) may provide the most effective and safest way of reversal. These agents are currently in premarketing studies.


Context: The aim of this study was to investigate the effect of inhibiting αvβ(3)/α(v) β(5) integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ(3) and αvβ(5) integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle-treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ(3)/αvβ(5) inhibition may be a promising therapeutic approach for bone metastases.


Context: With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4 mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4 mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4 mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4 mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.


Context: Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α- and β-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the 'defensin vaccine' concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.


Context: Seventeen children and young adults with Moebius syndrome were examined with a view to finding symptoms of autism. Some 40% of the group showed all or many of the symptoms typical of autistic disorder. The high frequency of autistic symptoms in Moebius syndrome might be a marked overrepresentation and could be suggestive of a common underlying neurobiological deficit at the brainstem level.


Context: Intercropping and rotating banana (Musa spp.) with Chinese chive (Allium tuberosum Rottler) has been used as an effective method to control Panama disease (Fusarium wilt) of banana in South China. However, the underlying mechanism is unknown. In this study, we used aqueous leachates and volatiles from Chinese chive to evaluate their antimicrobial activity on Fusarium oxysporum f. sp. cubense race 4 (FOC), the causal agent of Panama disease in banana, and identified the antifungal compounds. Both leaf and root leachates of Chinese chive displayed strong inhibition against FOC, but the concentrated leachates showed lower inhibition than the original leachates. In a sealed system volatiles emitted from the leaves and roots of Chinese chive inhibited mycelial growth of FOC. Volatile compounds emitted from the intact growing roots mimicking natural environment inhibited spore germination of FOC. We identified five volatiles including 2-methyl-2-pentenal and four organosulfur compounds (dimethyl trisulfide, dimethyl disulfide, dipropyl disulfide, and dipropyl trisulfide) from the leaves and roots of Chinese chive. All these compounds exhibited inhibitory effects on FOC, but 2-methyl-2-pentenal and dimethyl trisulfide showed stronger inhibition than the other three compounds. 2-Methyl-2-pentenal at 50-100 μl/l completely inhibited the mycelial growth of FOC. Our results demonstrate that antifungal volatiles released from Chinese chive help control Panama disease in banana. We conclude that intercropping and rotating banana with Chinese chive can control Panama disease and increase cropland biodiversity.


Context: Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.


Context: New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and in vivo studies to assess their safety and suitability for testing in humans. Regulatory health authorities require that therapeutic and supratherapeutic doses be administered, by the intended route of administration, to two nonclinical species prior to human testing (ICH Expert Working Group. The international conference on harmonization of technical requirements for registration of pharmaceuticals for human use (ICH); Multidisciplinary guidelines; Nonclinical safety studies (M3). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf , 2009). The purpose of these studies is to identify potential target organ toxicity and to determine if the effects are reversible. Liver is a potential site for toxicity caused by orally administered NMEs due to high exposure during first pass after oral administration. A range of clinical chemistry analytes are routinely measured in both nonclinical and clinical studies to evaluate and monitor for hepatotoxicity. While bilirubin itself circulates within a wide range of concentrations in many animal species and humans, without causing adverse effects and possibly providing benefits (Sedlak and Snyder. Pediatrics 113(6):1776-1782, 2004), bilirubin is one of the few readily monitored circulating biomarkers that can provide insight into liver function. Therefore, any changes in plasma or urine bilirubin levels must be carefully evaluated. Changes in bilirubin may occur as a result of adaptive nontoxic changes or severe toxicity. Examples of adaptive nontoxic changes in liver function, which may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport (Keogh. Adv Pharmacol 63:1-42, 2012). Alternatively, hepatocellular necrosis, hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some cases, these effects may be irreversible (FDA/CDER. Guidance for industry drug-induced liver injury: premarketing clinical evaluation. http://www.fda.gov/downloads/Drugs/…/Guidances/UCM174090.pdf , 2012).This chapter aims to demonstrate application of enzyme kinetic principles in understanding the risk of bilirubin elevation through inhibition of multiple processes-involving both enzymes and transporters. In the sections that follow, we first provide a brief summary of bilirubin formation and disposition. Two case examples are then provided to illustrate the enzyme kinetic studies needed for risk assessment and for identifying the mechanisms of bilirubin elevation. Caveats of methods and data interpretation are discussed in these case studies. The data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in enzyme kinetics to the reader.


Context: Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.


Context: During a 3-yr comprehensive study, 196 ixodid ticks (9 species) were collected from 89 passerine birds (32 species) from 25 localities across Canada to determine the distribution of avian-associated tick species and endogenous Lyme disease spirochetes, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner. We report the following first records of tick parasitism on avian hosts: the rabbit-associated tick, Ixodes dentatus Marx, from Manitoba and Ontario; the mouse tick, Ixodes muris Bishopp and Smith, from British Columbia; and the blacklegged tick, Ixodes scapularis Say, from New Brunswick. Moreover, we provide the first record of the Neotropical tick, Amblyomma humerale Koch (1 nymph), in Canada and its parasitism of any bird. This tick was compared morphologically with nymphs of other Neotropical Amblyomma spp., and genetically, using a 344-bp fragment of the 12S rDNA sequence of 41 New World Amblyomma species. The first collections of the western blacklegged tick, Ixodes pacificus Cooley and Kohls, from passerine species in Alberta and British Columbia, are also reported. Notably, we further report the first isolation of B. burgdorferi from the bird tick, Ixodes auritulus Neumann, collected from an American robin, Turdus migratorius L., on Vancouver Island. Furthermore, B. burgdorferi-positive I. auritulus larvae were collected from a reservoir-competent fox sparrow, Passerella iliaca (Merrem). Our findings indicate that ground-dwelling passerines, in particular, are parasitized by certain ixodid ticks and play an important role across Canada in the wide dispersal of B. burgdorferi-infected ticks and increased risk of Lyme disease exposure.


Context: Deficiency of the circadian clock transcriptional factor BMAL1 results in the development of premature aging in mice. In agreement with the accelerated aging phenotype, we observed an increase in the number of senescent cells in different tissues (lungs, liver and spleen) of Bmal1(-/-) mice, which suggests the important role of BMAL1 in the control of senescence in vivo. However, no difference in the rate of proliferation and senescence between primary fibroblasts isolated from wild-type and Bmal1(-/-) mice has been detected, suggesting that BMAL1 does not play a significant role in replicative senescence in vitro. BMAL1 deficient fibroblasts had an increased sensitivity to hydrogen peroxide treatment, and reduced sensitivity to DNA damaging anticancer drugs etoposide and daunorubicin. Increased sensitivity of Bmal1(-/-) cells to oxidative stress was p53 independent and correlated with the disrupted regulation of reactive oxygen species (ROS) homeostasis in BMAL1 deficient cells: indeed, circadian oscillations of ROS level can be induced in wild-type but not in Bmal1(-/-) cells. We propose that BMAL1 is important for the regulation of oxidative stress and DNA damage responses, while deregulation of these processes upon BMAL1 deficiency leads to development of stress induced senescence in vivo.


Context: Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. Here we employ quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in Drosophila embryos. We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We further reveal that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. We propose that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.


Context: As orthologous genes from related species diverge over time, some sequences are conserved in noncoding regions. In mammals, large phylogenetic footprints, or conserved noncoding sequences (CNSs), are known to be common features of genes. Here we present the first large-scale analysis of plant genes for CNSs. We used maize and rice, maximally diverged members of the grass family of monocots. 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. Grass genes have dramatically fewer and much smaller CNSs than mammalian genes. Twenty-seven percent of grass gene comparisons revealed no CNSs. Genes functioning in upstream regulatory roles, such as transcription factors, are greatly enriched for CNSs relative to genes encoding enzymes or structural proteins. Further, we show that a CNS cluster in an intron of the knotted1 homeobox gene serves as a site of negative regulation. We showthat CNSs in the adh1 gene do not correlate with known cis-acting sites. We discuss the potential meanings of CNSs and their value as analytical tools and evolutionary characters. We advance the idea that many CNSs function to lock-in gene regulatory decisions.


Context: Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII-dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in "backward" translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.


Context: Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of , the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease-causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the  gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO-mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with  mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression.


Context: In bacterial cells, bidirectional replication of the circular chromosome is initiated from a single origin (oriC) and terminates in an antipodal terminus region such that movement of the pair of replication forks is largely codirectional with transcription. The terminus region is flanked by discrete Ter sequences that act as polar, or direction-dependent, arrest sites for fork progression. Alternative oriC-independent modes of replication initiation are possible, one of which is constitutive stable DNA replication (cSDR) from transcription-associated RNA-DNA hybrids or R-loops. Here, I discuss the distinctive attributes of fork progression and termination associated with different modes of bacterial replication initiation. Two hypothetical models are proposed: that head-on collisions between pairs of replication forks, which are a feature of replication termination in all kingdoms of life, provoke bilateral fork reversal reactions; and that cSDR is characterized by existence of distinct subpopulations in bacterial cultures and a widespread distribution of origins in the genome, each with a small firing potential. Since R-loops are known to exist in eukaryotic cells and to inflict genome damage in G1 phase, it is possible that cSDR-like events promote aberrant replication initiation even in eukaryotes.


Context: Gastric cancer (GC) is a major world-wide health problem. It is the third leading cause of death from cancer. The treatment of advanced GC by chemotherapy has limited efficacy. The addition of some targeted therapies like trastuzumab and ramucirumab have added a modest benefit, but only in human epidermal growth factor receptor 2 (ERBB2 or HER2)-positive patients and in the second-line setting, respectively. The development of new and effective therapeutic strategies must consider the genetic complexity and heterogeneity of GC; prognostic and predictive biomarkers should be identified for clinical implementation. Immune deregulation has been associated with some GC subtypes, especially those that are associated with virus infection and those with a high mutational rate. Different mechanisms to prevent immunologic escape have been characterized during the last years; in particular the PD-1/PD-L1 inhibitors pembrolizumab, avelumab, durvalumab and atezolizumab have shown early sign of efficacy. Therefore, immunotherapeutic strategies may provide new opportunities for GC patients. This review will discuss (1) the main characteristics of GC treatment, (2) the immune response in GC, and (3) the current status of immune-related strategies in clinical development in GC patients, focusing on immune checkpoints therapies.


Context: Clinical questionWhat is the diagnostic accuracy of the PECARN, CATCH, and CHALICE clinical decision rules for pediatric head injury, and are the clinical decision rules valid when applied to a novel data set?Article chosenBabl FE, Borland ML, Phillips N, et al. Accuracy of PECARN, CATCH, and CHALICE head injury decision rules in children: a prospective cohort study. Lancet (London, England) 2017;389(10087):2393-402. OBJECTIVES: The primary objective of the study was to determine the diagnostic accuracy and provide external validation for the PECARN, CATCH, and CHALICE clinical decision rules in a clinically homogeneous cohort of children. The secondary objective of this study was to perform a direct comparison of the three decision rules by assessing for the presence of traumatic brain injury (TBI) on computed tomography (CT) or the requirement for neurointervention.


Context: Extensive histological and immunohistochemical studies were performed to elucidate the histopathogenesis of exencephaly induced in chick embryo as an experimental model. The findings were compared with those identified in a chick myeloschisis experimental model and in human autopsy cases. The experimental model of exencephaly in chick embryos was developed by induction with various teratogens including ethylnitrosourea, salicylate, and phenytoin. None of the cases of exencephaly was exposed to a teratogen prior to or within Hamburger and Hamilton stage 12 (45 to 49 hours postincubation), when the anterior neuropore closes. The process of overgrowth in development of exencephaly was identical to that of myeloschisis, and the results suggested neuronal overmaturation in the histological and immunohistochemical studies. Although the late-stage degenerative change with neovascularization over the exposed neural tissue (placode) was more severe in human exencephaly, the present experimental study may suggest a possible common embryopathogenesis of dysraphism. Exencephaly should be regarded as the most severe form of cranium bifidum, as myeloschisis is in spina bifida.


Context: T-cell malignancies are rare, making up 10% to 15% of all lymphoid neoplasms in adults. They include many different types of disorders such as T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, adult T-cell leukemia/lymphoma, cutaneous T-cell lymphoma, and peripheral T-cell lymphoma, which are themselves divided into multiple subcategories. Most T-cell malignancies arise as a result of chromosomal abnormalities, including T-cell receptor rearrangement anomalies. Viral infections are implicated in the development of adult T-cell leukemia/lymphoma and some cases of peripheral T-cell lymphoma have been linked to Epstein-Barr virus or human immunodeficiency virus infection. With the possible exception of T-cell large granular lymphocytic leukemia, which often has an indolent course, T-cell malignancies have not responded well to conventional chemotherapeutic treatment. The introduction of monoclonal antibodies for the treatment of cancer has changed the outlook for patients with T-cell malignancies. Recent studies with single-agent alemtuzumab, an anti-CD52 monoclonal antibody, have shown improved response rates and survival in patients with T-cell prolymphocytic leukemia and cutaneous T-cell lymphoma. Preliminary data also suggest that alemtuzumab may have activity in patients with heavily pretreated peripheral T-cell lymphoma who are refractory to conventional chemotherapy. Preclinical studies with mice bearing human adult T-cell leukemia/lymphoma cells suggest that alemtuzumab may have a potential therapeutic role in this setting. Treatment of T-cell hematologic malignancies with alemtuzumab appears promising. Earlier treatment and combination with chemotherapeutic agents may improve treatment outcome for patients with these malignancies and allow for consolidation with stem cell transplant strategies in selected patients.


Context: Saposin C is one of four homologous proteins derived from sequential cleavage of the saposin precursor protein, prosaposin. It is an essential activator for glucocerebrosidase, the enzyme deficient in Gaucher disease. Gaucher disease is a rare autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene that exhibits vast phenotypic heterogeneity, despite its designation as a "simple" Mendelian disorder. The observed phenotypic variability has led to a search for disease modifiers that can alter the Gaucher phenotype. The PSAP gene encoding saposin C is a prime candidate modifier for Gaucher disease. In humans, saposin C deficiency due to mutations in PSAP results in a Gaucher-like phenotype, despite normal in vitro glucocerebrosidase activity. Saposin C deficiency has also been shown to modify phenotype in one mouse model of Gaucher disease. The role of saposin C as an activator required for normal glucocerebrosidase function, and the consequences of saposin C deficiency are described, and are being explored as potential modifying factors in patients with Gaucher disease.


Context: Psoriasis is a chronic, genetically determined, immune-mediated, inflammatory skin disease affecting approximately 2% to 3% of Caucasian population. Given the well-established role of the immuno-mediated inflammation in the pathogenesis of psoriasis, in the past few years several key steps in the pathogenesis of this disease have been elucidated and the increased knowledge led to the development of specific drugs, commonly defined as "biologics" targeting one or more of these steps. At present an anti-CD11a antibody (efalizumab), an anti-LFA3/CD2 receptor (alefacept) and 3 antitumor necrosis factor alpha agents (adalimumab, etanercept, infliximab) are now commercially available for the treatment of both psoriasis and psoriatic arthritis. Recent studies have demonstrated that interleukins (IL) 12 and 23 play an important role in the pathophysiology of psoriasis. In fact members of the IL-12 family of cytokines have the potential to act as the next major cytokine(s) in pathogenesis and the treatment of psoriasis. Ustekinumab (CNTO 1275, Centocor Inc, Malvern, PA, USA) is a human monoclonal antibody that binds to the shared p40 protein subunit of human interleukins 12 and 23 with high affinity and specificity, thereby preventing interaction with their surface IL-12Rβ1 receptor. Different clinical studies have been conducted to date. In particular a phase II study and two phase III studies, PHOENIX 1 together with PHOENIX 2, show very encouraging results. This review reports on the latest progress made in the clinical use of biologic drugs for psoriasis focusing on the new human IL-12/23 monoclonal antibody, ustekinumab, for psoriasis.


Context: Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.


Context: Given the increasing costs of pharmaceuticals today, it is important to understand how pharmacy benefits decisions are made and the role of cost and values in these decisions. This study examines what coverage decisions insurers make and the information and processes used in making these decisions. Fifty-three organizations, differing in size, tax status, and region, were asked about their policies for four new and controversial drugs: Viagra, Enbrel, Zyban, and Celebrex. Enbrel and Celebrex were much more likely to be covered than Viagra and Zyban. In addition, coverage of Enbrel and Celebrex was limited through strategies such as prior authorization, to encourage medically appropriate use of these agents, whereas coverage of Viagra and Zyban was limited predominantly through generalized exclusion or through restrictions on quantity or duration of use. Value judgments, rather than cost, seem to play a central, though largely unspoken, role in these coverage decisions.


Context: The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.


Context: Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses.


Context: These data indicate that T(3) replacement to euthyroid levels improves systolic function and tends to improve diastolic function, potentially through changes in myocardial gene expression.


Context: Social recognition memory is an essential and basic component of social behavior that is used to discriminate familiar and novel animals/humans. Previous studies have shown the importance of several brain regions for social recognition memories; however, the mechanisms underlying the consolidation of social recognition memory at the molecular and anatomic levels remain unknown. Here, we show a brain network necessary for the generation of social recognition memory in mice. A mouse genetic study showed that cAMP-responsive element-binding protein (CREB)-mediated transcription is required for the formation of social recognition memory. Importantly, significant inductions of the CREB target immediate-early genes c-fos and Arc were observed in the hippocampus (CA1 and CA3 regions), medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and amygdala (basolateral region) when social recognition memory was generated. Pharmacological experiments using a microinfusion of the protein synthesis inhibitor anisomycin showed that protein synthesis in these brain regions is required for the consolidation of social recognition memory. These findings suggested that social recognition memory is consolidated through the activation of CREB-mediated gene expression in the hippocampus/mPFC/ACC/amygdala. Network analyses suggested that these four brain regions show functional connectivity with other brain regions and, more importantly, that the hippocampus functions as a hub to integrate brain networks and generate social recognition memory, whereas the ACC and amygdala are important for coordinating brain activity when social interaction is initiated by connecting with other brain regions. We have found that a brain network composed of the hippocampus/mPFC/ACC/amygdala is required for the consolidation of social recognition memory. Here, we identify brain networks composed of multiple brain regions for the consolidation of social recognition memory. We found that social recognition memory is consolidated through CREB-meditated gene expression in the hippocampus, medial prefrontal cortex, anterior cingulate cortex (ACC), and amygdala. Importantly, network analyses based on c-fos expression suggest that functional connectivity of these four brain regions with other brain regions is increased with time spent in social investigation toward the generation of brain networks to consolidate social recognition memory. Furthermore, our findings suggest that hippocampus functions as a hub to integrate brain networks and generate social recognition memory, whereas ACC and amygdala are important for coordinating brain activity when social interaction is initiated by connecting with other brain regions.


Context: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary cerebral angiopathy. We present a case in which a pattern of diffusion signal change compatible with bihemispheric acute watershed infarcts occurred in a symptomatic patient demonstrating global hypoperfusion. To our knowledge, watershed infarcts in the clinical presentation of CADASIL have not been previously described.


Context: The clinical presentation of dural arteriovenous fistulas (DAVFs), in particular the associated risk of intracranial hemorrhage, shows a strong correlation with their pattern of venous drainage. The two most commonly used and clinically accepted DAVF classifications are the Merland-Cognard classification and the Borden classification, both based on the morphology of the venous drainage. A revised classification that grades DAVFs through a combination of angiographic and clinical features has also been proposed. This article offers a review of these various classification schemes, and discusses their application to treatment decision making.


Context: The optimal treatment strategy for anaplastic oligodendroglial (AO) tumors is evolving. Molecular profiling of oligodendrogliomas have shown distinctive genetic patterns characterized by combined deletions of chromosome arms 1p and 19q, O(6)-methylguanine methyltransferase (MGMT) methylation, and isocitrate dehydrogenase 1 (IDH1) mutations; they are all prognostic factors for patients with AO. In addition, a strong association has also been found between the CpG island hypermethylation phenotype (CIMP) status and MGMT promoter methylation. Long term follow up data of the Radiation Therapy Oncology Group (RTOG) 9402 and the European Organisation for Research and Treatment of Cancer (EORTC) 26951 studies demonstrate clear evidence that for patients with codeleted 1p19q AO, early chemotherapy with radiation offers a significant improvement in overall survival compared with early radiation, even with salvage chemotherapy at tumor relapse, and thus establishes the 1p19q allelic loss as a predictive marker distinct from tumors without the chromosome change. Radiotherapy alone is no longer considered an adequate treatment for this patient population. In cases with no 1p19q deletion, most neuro-oncologists recommend incorporating radiotherapy into the upfront treatment strategy. However, there are still unanswered questions regarding whether upfront chemotherapy, omitting/deferring radiotherapy, in the desire to avoid late neurocognitive toxicity of radiotherapy should be the initial therapy for AO tumors with codeleted 1p19q, or whether temozolomide, an oral agent with a better toxicity profile, can be substituted for procarbazine, lomustine, and vincristine (PCV). Further studies are warranted and the increasing understanding of molecular pathways involved may lead to more selective therapeutic targets in the future.


Context: 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.


Context: Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.


Context: Lasmiditan is a novel selective 5-HT(1F) receptor agonist. It is both scientifically and clinically relevant to review whether a 5-HT(1F) receptor agonist is effective in the acute treatment of migraine. Two RCTs in the phase II development of lasmiditan was reviewed. In the intravenous placebo-controlled RCT, lasmiditan doses of 2.5-45 mg were used, and there was a linear association between headache relief (HR) rates and dose levels (P < 0.02). For lasmiditan 20 mg, HR was 64 % and for placebo it was 45 % (NS). In the oral placebo-controlled RCT, lasmiditan doses of 50, 100, 200 and 400 mg were used. For HR, all doses of lasmiditan were superior to placebo (P < 0.05). For lasmiditan 400 mg, HR was 64 % and it was 25 % for placebo. Adverse events (AEs) emerging from the treatment were reported by 22 % of the patients receiving placebo and by 65, 73, 87 and 87 % of patients receiving 50, 100, 200 and 400 mg, respectively. The majority of AEs after lasmiditan 100 and 400 mg were moderate or severe. For the understanding of migraine pathophysiology, it is very important to note that a selective 5-HT(1F) receptor agonist like lasmiditan is effective in the acute treatment of migraine. Thus, migraine can be treated with a drug that has no vasoconstrictor ability. While lasmiditan most likely is effective in the treatment of migraine attacks it had, unfortunately, a high incidence of CNS related AEs in the oral RCT. If confirmed in larger studies in phase III, this might adversely limit the use of this highly specific non-vascular acute treatment of migraine. Larger studies including the parameters of patients' preferences are necessary to accurately position this new treatment principle in relation to the triptans.


Context: Hypoglycin A, the toxin found in the ackee fruit, has been reported in the literature as the causative agent in incidences of acute toxicity termed Jamaican vomiting sickness or toxic hypoglycemic syndrome. Hypoglycin A toxicity in this study was determined by feeding male and female Sprague-Dawley rats a control diet and ackee diets that contained 4-3840 ppm of hypoglycin. The fixed dose method was used to quantify the acute toxic dose of hypoglycin A and was determined by feeding a diet consisting of the lowest hypoglycin A concentration; this was increased to the next highest dose after 24h until toxicity was observed. The maximum tolerated dose (MTD) of hypoglycin A was determined by feeding rats the ackee and control diets over a 30-day period. The acute toxic dose for male and female rats was 231.19+/-62.5 5mg hypoglycinA/kgBW and 215.99+/-63.33 mg hypoglycinA/kgBW, respectively. This was considerably greater than the dose of 100 mg hypoglycin/kgBW reported in a previous study when aqueous hypoglycin was administered orally. The MTD of hypoglycin A in both male and female rats was 1.50+/-0.07 mg hypoglycinA/kgBW/day. These findings suggest that the form in which hypoglycin in ackee is administered could affect the toxicological properties it exhibits. Therefore, for the purpose of a hazard assessment, it may be best administered within the matrix of the fruit, which is the form that humans consume it.


Context: We have previously shown that acute thyroid hormone treatment could limit reperfusion injury and increase post-ischemic recovery of function. In the present study, we further explore potential initiating mechanisms of this response. Thus, isolated rat hearts were subjected to 30 min zero-flow global ischemia (I) followed by 60-min reperfusion (R). Reperfusion injury was assessed by post-ischemic recovery of left ventricular developed pressure (LVDP%) and LDH release. T3 at a dose of 60 nM which had no effect on contractile function of non-ischemic myocardium, significantly increased LVDP% [48% (2.9) vs. 30.2% (3.3) for untreated group, P < 0.05] and reduced LDH release [8.3 (0.3) vs. 10 (0.42) for untreated group, P < 0.05] when administered at R. T4 (60 and 400 nM) had no effect on contractile function either in non-ischemic or ischemic myocardium. Administration of debutyl-dronedarone (DBD), a TRα1 antagonist abolished the T3-limiting effect on reperfusion injury: Thus, co-administration of T3 and DBD resulted in significantly lower LVDP%, [23% (4.7) vs. 48% (2.9) for T3 group, P < 0.05] and higher LDH release [9.9 (0.3) vs. 8.3 (0.3), for T3 group, P < 0.05]. In conclusion, acute T3 and not T4 treatment will be able to protect against reperfusion injury. T3 can exert this beneficial effect on ischemic myocardium at a dose that has no effects on non-ischemic myocardium. Acute T3-limiting effect on reperfusion injury is mediated, at least in part, via TRα1 receptor.


Context: Triptolide is a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. Recent studies show that T cells play an important role in neuropathic pain following nerve injury in rats. In this study, we investigated the effect of triptolide on T cell activation and development of neuropathic pain. Neuropathic pain by chronic constriction injury (CCI) was induced by loose ligation of the sciatic nerve in Sprague-Dawley rats. Triptolide (5 or 10 μg/kg) or vehicle (DMSO) was administered intrathecally after surgery for 7 days (n=8 per group). The right hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before and after the surgery (days 0 to 7). NF-κB activation and pro-inflammatory cytokine (TNF-α and IL-2) expression were determined by ELISA, Western blot, and real time-PCR. CCI of the sciatic nerve induced mechanical allodynia and thermal hyperalgesia in these rats. Intrathecal triptolide (5 and 10 μg/kg) suppressed the development of allodynia and thermal hyperalgesia. It also inhibited CCI-induced inflammation and T cell activation, by decreasing spinal cord TNF-α, IL-2 and NF-κB p65 levels. Motor dysfunction was not observed after triptolide treatment. In the present study, we demonstrated the suppressive effect of triptolide on the development of neuropathic pain. Therefore, triptolide could be a promising immunosuppressive agent in the treatment of neuropathic pain. Further studies are required to examine the safety of intrathecal triptolide for clinical application.


Context: There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation and can occur selectively for substrates, including aggregated proteins. It remains unclear, however, whether starvation-induced and selective macroautophagy share identical adaptor molecules to capture their cargo. Here, we report that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L, and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner and, likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy by bridging cargo to the molecular machinery that builds autophagosomes.


Context: Ubiquilins (UBQLN), a family of adaptor proteins with partial homology with ubiquitin, are proposed to facilitate proteasomal degradation of ubiquitinated substrates. We now demonstrate a novel role for UBQLN in promoting autophagosome maturation during nutrient deprivation. Ectopic expression of UBQLN protects cells against starvation-induced cell death, while depletion renders cells more susceptible. This protective function requires the essential autophagy regulators, Atg5 and Atg7. The ubiquitin-associated (UBA) domain of UBQLN is required for its association with autophagosomes as well as for its prosurvival functions.Remarkably, during starvation-induced autophagy, UBQLN promotes the fusion of early autophagosomes with lysosomes.Overall, this work illustrates an important function for UBQLN in cell survival during nutrient starvation, which requires a newly recognized function for UBQLN in autophagosome maturation.


Context: Vedolizumab is a humanized monoclonal antibody that targets the alpha(4)beta(7) integrin exclusively, and modulates inflammation in the gastrointestinal tract without inducing the systemic immunosuppression that characterizes anti-alpha(4) chain monoclonal antibodies, such as natalizumab. This unique pharmacologic profile is largely attributable to four determinants. The first determinant is the restriction of the expression of the alpha(4)beta(7) integrin to subsets of leukocytes. Vedolizumab does not bind to the majority of memory CD4(+) T lymphocytes (60%), neutrophils, and most monocytes. The highest level of vedolizumab binding is to a subset (approximately 25%) of human peripheral blood memory CD4(+) T lymphocytes that include gut-homing interleukin 17 T-helper lymphocytes. Vedolizumab also binds to eosinophils at high levels, and to naive T-helper lymphocytes, naive and memory cytotoxic T lymphocytes, B lymphocytes, natural killer cells, and basophils at lower levels; vedolizumab binds to memory CD4(+) T and B lymphocytes with subnanomolar potency (EC(50) = 0.3-0.4 nM). The second determinant is binding specificity; vedolizumab binds exclusively to the alpha(4)beta(7) integrin, and not to the alpha(4)beta(1) and alpha(E)beta(7) integrins. The third determinant is selective antagonism; vedolizumab selectively inhibits adhesion of alpha(4)beta(7)-expressing cells to mucosal addressin cell adhesion molecule 1 (median inhibition concentration [IC(50)] = 0.02-0.06 microg/ml) and fibronectin (IC(50) = 0.02 microg/ml), but not vascular cell adhesion molecule 1. The fourth determinant is the gastrointestinal-specific tropism of the alpha(4)beta(7) integrin function. These pharmacologic properties of vedolizumab, in conjunction with the gastrointestinal tropism of alpha(4)beta(7) integrin function, may ultimately confer an improved risk-to-benefit profile for patients with inflammatory bowel diseases.


Context: The adipocytokine apelin is a peptide, Apelin and its receptor are abundantly expressed in the nervous and cardiovascular systems. Previous studies had found apelin-13 reduces brain injuries and postischemic cerebral edema through blocking programmed cell death, Apelin-13 is also able to inhibit glucose deprivation induced cardiomyocyte autophagy in a concentration dependent fashion. To observe the effect of Apelin-13 on the brain injury induced by traumatic brain injury (TBI), and explore the effect of Apelin-13 on autophagy in TBI, We performed The neurological test, and the numbers of TBI-induced neural cell death were also counted by propidium iodide labeling. At last, the autophagy associated proteins LC3, Beclin-1, Bcl-2, p62 were also assessed with western-blotting. Compared with saline vehicle groups, the neural cell death, lesion volume, and neural dysfunction were attenuated by apelin-13 after TBI. In additionally, Apelin-13 also reversed TBI induced downregulation of LC3, Beclin-1, Bcl-2, p62 expression, compared with saline vehicle groups, at 24 and 48 h post TBI. Apelin-13 attenuates TBI induced brain damage by suppressing autophagy. All these results revealed that Apelin-13 suppressed autophagy. The autophagy may be involved in the mechanism of Apelin-13 rescue the subsequent damaged neuron in TBI.


Context: Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.


Context: T cell DNA methylation levels decline with age, activating genes such as KIR and TNFSF7 (CD70), implicated in lupus-like autoimmunity and acute coronary syndromes. The mechanisms causing age-dependent DNA demethylation are unclear. Maintenance of DNA methylation depends on DNA methyltransferase 1 (Dnmt1) and intracellular S-adenosylmethionine (SAM) levels, and is inhibited by S-adenosylhomocysteine (SAH). SAM levels depend on dietary micronutrients including folate and methionine. SAH levels depend on serum homocysteine concentrations. T cell Dnmt1 levels also decline with age. We hypothesized that age-dependent Dnmt1 decreases synergize with low folate, low methionine or high homocysteine levels to demethylate and activate methylation-sensitive genes. T cells from healthy adults ages 22-81, stimulated and cultured with low folate, low methionine, or high homocysteine concentrations showed demethylation and overexpression of KIR and CD70 beginning at age approximately 50 and increased further with age. The effects were reproduced by Dnmt1 knockdowns in T cells from young subjects. These results indicate that maintenance of T cell DNA methylation patterns is more sensitive to low folate and methionine levels in older than younger individuals, due to low Dnmt1 levels, and that homocysteine further increases aberrant gene expression. Thus, attention to proper nutrition may be particularly important in the elderly.


Context: Advanced hepatocellular carcinoma (HCC) has limited treatment options and poor survival, therefore early detection is critical to improving the survival of patients with HCC. Current guidelines for high-risk patients include ultrasound screenings every six months, but ultrasounds are operator dependent and not sensitive for early HCC. Serum α-Fetoprotein (AFP) is a widely used diagnostic biomarker, but it has limited sensitivity and is not elevated in all HCC cases so, we incorporate a second blood-based biomarker, des'γ carboxy-prothrombin (DCP), that has shown potential as a screening marker for HCC. The data from the Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C) Trial is a valuable source of data to study biomarker screening for HCC. We assume the trajectories of AFP and DCP follow a joint hierarchical mixture model with random changepoints that allows for distinct changepoint times and subsequent trajectories of each biomarker. The changepoint indicators are jointly modeled with a Markov Random Field distribution to help detect borderline changepoints. Markov chain Monte Carlo methods are used to calculate posterior distributions, which are used in risk calculations among future patients and determine whether a patient has a positive screen. The screening algorithm was compared to alternatives in simulations studies under a range of possible scenarios and in the HALT-C Trial using cross-validation.


Context: Multiple myeloma (MM) is a disease of unknown, complex etiology that affects primarily older adults. The course of the disease and the patients' survival time are very heterogeneous, but over the last decade, clear progress in the treatment of this incurable disease has been observed. Therapeutics that have proven to be highly effective include the immunomodulatory drug thalidomide and its newer analogs, lenalidomide and pomalidomide, as well as the proteasome inhibitors bortezomib and carfilzomib. However, the administration of some of the treatments, e.g., thalidomide or bortezomib, has also been associated with the occurrence of a serious and common adverse effect, drug-induced peripheral neuropathy. The mechanism of the development of the peripheral neuropathy is poorly understood. Nevertheless, one of its potential causes could be inadequate concentrations of crucial trophic factors, including neurotrophic and/or angiogenic factors, which are responsible for the proliferation, differentiation, survival and death of neuronal and nonneuronal cells.


Context: Messenger RNA 3' end formation is an integral step in the process that gives rise to mature, translated messenger RNAs in eukaryotes. With this step, a pre-messenger RNA is processed and polyadenylated, giving rise to a mature mRNA bearing the characteristic poly(A) tract. The poly(A) tract is a fundamental feature of mRNAs, participating in the process of translation initiation and being the focus of control mechanisms that define the lifetime of mRNAs. Thus messenger RNA 3' end formation impacts two steps in mRNA biogenesis and function. Moreover, mRNA 3' end formation is something of a bridge that integrates numerous other steps in mRNA biogenesis and function. While the process is essential for the expression of most genes, it is also one that is subject to various forms of regulation, such that both quantitative and qualitative aspects of gene expression may be modulated via the polyadenylation complex. In this review, the current status of understanding of mRNA 3' end formation in plants is discussed. In particular, the nature of mRNA 3' ends in plants is reviewed, as are recent studies that are beginning to yield insight into the functioning and regulation of plant polyadenylation factor subunits.


Context: Ultraviolet radiation (UVR) exposure causes various injurious effects to human skin by generating reactive oxygen species (ROS). Excessive ROS production can lead to oxidative stress which may damage cellular components like lipids and proteins and causing photoaging. The use of natural photochemopreventive agents with antioxidant properties is an important alternative to improve the effectiveness of sunscreens and reduce skin photodamage. A crude extract (CE) from the leaves of Arrabidaea chica underwent partition by a liquid-liquid method. The hexane fraction (FH), chloroform fraction (FC), and ethyl acetate fraction (FEA) were obtained. The antioxidant capacity of the CE, FH, FC, and FEA was studied in a cell-free system using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and the xanthine/luminol/xanthine oxidase system. The FC had the best antioxidant activity. We also evaluated the photochemoprotective effect of A. chica in protecting L929 fibroblasts against UV-A- and UV-B-induced cell damage. A. chica inhibited the extended production of ROS up to 3h. Posttreatment with the CE and FC attenuated UV-induced cell damage through scavenging mechanisms, including the quenching of intracellular ROS and mitochondrial O and preventing lipid peroxidation. These results suggest that A. chica may be a promising non-sunscreen photoprotector that can improve the effectiveness of commercial sunscreens.


Context: Despite the known effectiveness of anti-inflammatory therapy in reducing reperfusion injury, no studies to date involve the use of anti-inflammatory therapy in reducing ischemia-reperfusion injury in fasciocutaneous flaps. Dexamethasone (a phospholipase A2 inhibitor) and specific cyclooxygenase and lipoxygenase inhibitors (indomethacin and BW755C) were administered to rats with ischemic island groin (fasciocutaneous) flaps. Significant improvement in ischemic flap survival was found with dexamethasone and BW755C. The mode of action of dexamethasone was not specifically investigated in our study; however, it may suppress neutrophil function and reduce ischemia-reperfusion injury in its shared ability with BW755C to reduce the formation of leukotrienes. Dexamethasone could be applied in the clinical setting to reduce ischemic flap loss by attenuating the systemic inflammatory response to reperfused ischemic-damaged tissue.


Context: Klinefelter's syndrome is one of the most common forms of primary hypogonadism and infertility in males. It is characterized by small and firm testes, gynecomastia, azoospermia, and an elevated gonadotropin level. The frequencies of diabetes mellitus, breast cancer, and germ cell neoplasia increases in Klinefelter's syndrome. We report upon a 35 year-old male patient with Graves' disease in association with Klinefelter's syndrome; as confirmed by chromosome analysis. The patient is being treated with antithyroid medication for Graves' disease and by testosterone replacement for Klinefelter's syndrome.


Context: Our understanding of the molecular pathogenesis of the neuromuscular ion channelopathies has increased rapidly over the past two decades due to the identification of many of the genes whose mutation causes these diseases. These molecular discoveries have facilitated identification and classification of the hereditary periodic paralyses and the myotonias, and are likely to shed light on acquired ion channelopathies as well. Despite our better understanding of the pathogenesis of these disorders, current treatments are largely empirical and the evidence in favor of specific therapy largely anecdotal. For periodic paralysis, dichlorphenamide--a carbonic anhydrase inhibitor--has been shown in a controlled trial to prevent attacks for many patients with both hypokalemic and hypokalemic periodic paralysis. A second trial, comparing dichlorphenamide with acetazolamide versus placebo, is currently in progress. For myotonia, there is only anecdotal evidence for treatment, but a controlled trial of mexiletine versus placebo is currently being funded by a Food and Drug Administration-orphan products grant and is scheduled to begin in late 2008. In the future, mechanism-based approaches are likely to be developed. For example, exciting advances have already been made in one disorder, myotonic dystrophy-1 (DM-1). In a mouse model of DM-1, a morpholino antisense oligonucleuotide targeting the 3' splice site of CLCN1 exon 7a repaired the RNA splicing defect by promoting the production of full-length chloride channel transcripts. Abnormal chloride conductance was restored, and myotonia was abolished. Similar strategies hold potential for DM-2. The era of molecularly-based treatments is about to begin.


Context: Transcription factor FOXP3 (forkhead box P3) is found initially as a key regulator in regulatory T cells. Recently its expression has been demonstrated in some non-lymphoid normal and cancerous cells. Now FOXP3 has been proven to regulate cancer-related genes, especially suppressor genes in breast cancer. But the mechanisms by which FOXP3 regulates suppressor genes are not fully determined. In this study, we found the inverse correlation between FOXP3 and Ezh2, an enzyme for histone H3K27 trimethylation (H3K27me3) and a central epigenetic regulator in cancer. The overexpression of FOXP3 weakened Ezh2's enhancement on the mammosphere formation, cell proliferation, directional migration, and colony forming ability of T47D cells. We demonstrated that FOXP3 could downregulate Ezh2 protein level and this depended on not only the FOXP3 expression amount, but also the nuclear localization of FOXP3. More importantly, we demonstrated FOXP3 accelerated Ezh2 protein degradation through the polyubiquitination-proteasome pathway by enhancing the transcription of E3 ligase Praja1 directly. These results provided a new mechanism for FOXP3 in histone modifications as an Ezh2 suppressor and supported new evidence for FOXP3 as a tumor suppressor in breast cancer.


Context: Gabapentin gives moderate results in children with OAB refractory to conventional anticholinergics. In general, the drug is well tolerated with fewer adverse effects.


Context: Seventy-four Fusarium oxysporum soil isolates were assayed for known effector genes present in an F. oxysporum f. sp. lycopersici race 3 tomato wilt strain (FOL MN-25) obtained from the same fields in Manatee County, Florida. Based on the presence or absence of these genes, four haplotypes were defined, two of which represented 96% of the surveyed isolates. These two most common effector haplotypes contained either all or none of the assayed race 3 effector genes. We hypothesized that soil isolates with all surveyed effector genes, similar to FOL MN-25, would be pathogenic toward tomato, whereas isolates lacking all effectors would be nonpathogenic. However, inoculation experiments revealed that presence of the effector genes alone was not sufficient to ensure pathogenicity on tomato. Interestingly, a nonpathogenic isolate containing the full suite of unmutated effector genes (FOS 4-4) appears to have undergone a chromosomal rearrangement yet remains vegetatively compatible with FOL MN-25. These observations confirm the highly dynamic nature of the F. oxysporum genome and support the conclusion that pathogenesis among free-living populations of F. oxysporum is a complex process. Therefore, the presence of effector genes alone may not be an accurate predictor of pathogenicity among soil isolates of F. oxysporum.


Context: Onartuzumab, a humanized, monovalent monoclonal anti-MET antibody, antagonizes MET signaling by inhibiting binding of its ligand, hepatocyte growth factor (HGF). We investigated the effects of onartuzumab on cell-associated and circulating (shed) MET (sMET) and circulating HGF in vitro and nonclinically to determine their utility as pharmacodynamic biomarkers for onartuzumab. Effects of onartuzumab on cell-associated MET were assessed by flow cytometry and immunofluorescence. sMET and HGF were measured in cell supernatants and in serum or plasma from multiple species (mouse, cynomolgus monkey, and human) using plate-based immunoassays. Unlike bivalent anti-MET antibodies, onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. Onartuzumab delayed the clearance of human xenograft tumor-produced sMET from the circulation of mice, and endogenous sMET in cynomolgus monkeys. In mice harboring MET-expressing xenograft tumors, in the absence of onartuzumab, levels of human sMET correlated with tumor size, and may be predictive of MET-expressing tumor burden. Because binding of sMET to onartuzumab in circulation resulted in increasing sMET serum concentrations due to reduced clearance, this likely renders sMET unsuitable as a pharmacodynamic biomarker for onartuzumab. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or endogenous HGF in cynomolgus monkeys. Although sMET and HGF may serve as predictive biomarkers for MET therapeutics, these data do not support their use as pharmacodynamic biomarkers for onartuzumab.


Context: An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.


Context: This review critically analyzes how machine learning is being used to support clinical decision-making in the management of potentially resectable pancreatic cancer. Following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines, electronic searches of MEDLINE, Embase, PubMed, and Cochrane Database were undertaken. Studies were assessed using the checklist for critical appraisal and data extraction for systematic reviews of prediction modeling studies (CHARMS) checklist. In total 89,959 citations were retrieved. Six studies met the inclusion criteria. Three studies were Markov decision-analysis models comparing neoadjuvant therapy versus upfront surgery. Three studies predicted survival time using Bayesian modeling (n = 1) and artificial neural network (n = 1), and one study explored machine learning algorithms including Bayesian network, decision trees, k-nearest neighbor, and artificial neural networks. The main methodological issues identified were limited data sources, which limits generalizability and potentiates bias; lack of external validation; and the need for transparency in methods of internal validation, consecutive sampling, and selection of candidate predictors. The future direction of research relies on expanding our view of the multidisciplinary team to include professionals from computing and data science with algorithms developed in conjunction with clinicians and viewed as aids, not replacement, to traditional clinical decision-making.


Context: The use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS(n)) for the glycoproteomic characterization of glycopeptides is a growing field of research. The N- and O-glycosylated peptides (N- and O-glycopeptides) analyzed typically originate from protease-digested glycoproteins where many of them are expected to be biomedically important. Examples of LC-MS(2) and MS(3) fragmentation strategies used to pursue glycan structure, peptide identity and attachment-site identification analyses of glycopeptides are described in this review. MS(2) spectra, using the CID and HCD fragmentation techniques of a complex biantennary N-glycopeptide and a core 1 O-glycopeptide, representing two examples of commonly studied glycopeptide types, are presented. A few practical tips for accomplishing glycopeptide analysis using reversed-phase LC-MS(n) shotgun proteomics settings, together with references to the latest glycoproteomic studies, are presented.


Context: Insulin plays a very important role in maintaining homeostasis of the whole organism. It regulates glucose metabolism, glycogen synthesis, lipid and protein metabolism. Insulin receptors are present in virtually all cells, which is reflected by the diversity of regulatory processes in which this hormone is involved. Any dysfunction of insulin signalling pathway as a result of insulin receptor gene mutations is linked with various forms of insulin resistance, including insulin resistance type A, Donohue or Rabson-Mendenhall syndrome, which differ in the level of severity. Molecular analysis of insulin receptor gene may lead to a better understanding of molecular mechanisms underlying various types of insulin resistance and help to develop a more efficient treatment. They may also be used as a powerful tool in prenatal diagnostics as well as in pregnancy planning.


Context: Sotos syndrome is characterised by excessive pre and postnatal growth, a variable degree of learning difficulties and a recognisable facial appearance. This report highlights the difficulty in making the diagnosis where failure to thrive is the presenting feature and documents a previously undescribed association with recurrent parotitis.


Context: Tumor suppressor gene CYLD is a deubiquitinating enzyme which negatively regulates various signaling pathways by removing the lysine 63-linked polyubiquitin chains from several specific substrates. Loss of CYLD in different types of tumors leads to either cell survival or proliferation. In this study we demonstrate that lack of CYLD expression in CYLD-/- MEFs increases proliferation rate of these cells compared to CYLD+/+ in a serum concentration dependent manner without affecting cell survival. The reduced proliferation rate in CYLD+/+ in the presence of serum was due to the binding of serum response factor (SRF) to the serum response element identified in the CYLD promoter for the up-regulation of CYLD levels. The serum regulated recruitment of SRF to the CYLD promoter was dependent on p38 mitogen-activated protein kinase (MAPK) activity. Elimination of SRF by siRNA or inhibition of p38 MAPK reduced the expression level of CYLD and increased cell proliferation. These results show that SRF acts as a positive regulator of CYLD expression, which in turn reduces the mitogenic activation of serum for aberrant proliferation of MEF cells.


Context: Pyrimidine biosynthesis is an attractive drug target in a variety of organisms, including humans and the malaria parasite Plasmodium falciparum. Dihydroorotate dehydrogenase, an enzyme catalyzing the only redox reaction of the pyrimidine biosynthesis pathway, is a well-characterized target for chemotherapeutical intervention. In this study, we have applied SPROUT-LeadOpt, a software package for structure-based drug discovery and lead optimization, to improve the binding of the active metabolite of the anti-inflammatory drug leflunomide to the target cavities of the P. falciparum and human dihydroorotate dehydrogenases. Following synthesis of a library of compounds based upon the SPROUT-optimized molecular scaffolds, a series of inhibitors generally showing good inhibitory activity was obtained, in keeping with the SPROUT-LeadOpt predictions. Furthermore, cocrystal structures of five of these SPROUT-designed inhibitors bound in the ubiquinone binding cavity of the human dihydroorotate dehydrogenase are also analyzed.


Context: During the past three decades, the deleterious consequences of Chornobyl accident including carcinogenic effects in the people who were accidentally exposed to radiation have been intensively studied. In particular, recent studies provided increased knowledge of the molecular pathogenesis of thyroid tumors in children exposed to Chornobyl fallout. The risk of several forms of leukemia including myelodysplastic syndromes is elevated in Chornobyl liquidators. Furthermore, the upward trends of increases in a variety of other tumors including breast cancer, cancers of central nervous system and renal cancer have been reported in the persons exposed to Chornobyl fallout. There is growing evidence that insufficient apoptosis allows irradiated cells to survive and thereby contributes to carcinogenesis. The purpose of the present survey is to summarize the recent findings related to apoptotic biomarkers among cancer patients from the different populations affected by the Chornobyl catastrophe. Among the particularly radiosensitive cancer sites, we focused on thyroid cancer and leukemia. Several genes and/or proteins controlling apoptosis directly or indirectly have been incorporated into the analysis. The data reviewed here provide a mechanistic link between the apoptosis alterations and development of radiation-related cancer in the 30-year post-Chornobyl period. We suggest that the type of mutations arising from misrepair of DNA double strand breaks (gene fusion and amplification) is the initial signature event in radiation-induced thyroid cancer. Much work has to be done over the next years to elucidate central questions related to the nature of human radiation carcinogenesis. This article is part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".


Context: Neurofibroma is a benign tumor that arises from small or large nerves. This neoplastic lesion is a common feature of neurofibromatosis type 1 (NF1), one of the most common autosomal dominant disorders. The NF1 gene codes for a protein called "neurofibromin." It possesses a region that shares a high homology with the family of GTPase-activating proteins, which are negative regulators of RAS function and thereby control cell growth and differentiation. The evidence points to the NF1 gene being a tumor-suppressor gene. NF1 patients also have an increased incidence of certain malignant tumors that are believed to follow the "two hit" hypothesis, with one allele constitutionally inactivated and the other somatically mutated. Recently, somatic loss of heterozygosity (LOH) has been described for neurofibromas, and mutations in both copies of the NF1 gene have been reported for a dermal neurofibroma. The aim of our study was the analysis of the NF1 locus in benign neurofibromas in NF1 patients. We performed LOH analysis on 60 neurofibromas belonging to 17 patients, 9 of them with family history of the disease and 8 of them sporadic. We have analyzed five intragenic NF1 markers and six extragenic markers, and we have found LOH in 25% of the neurofibromas (corresponding to 53% of the patients). In addition, we found that in the neurofibromas of patients from familial cases the deletions occurred in the allele that is not transmitted with the disease, indicating that both copies of the NF1 gene were inactivated in these tumors. Therefore, the recent reports mentioned above, together with our findings, strongly support the double inactivation of the NF1 gene in benign neurofibromas.


Context: The present study describes a newly identified protein named CReMM (chromatin-related mesenchymal modulator). The protein was studied by bioinformatic means and classified as a member of the third subfamily of chromodomain helicase DNA-binding proteins (CHD). In silico translation defined CReMM as a multiple domains protein including two chromodomains, SNF2/ATPase, helicase C domain and an A/T-DNA-binding domain (DBD). Predicted extensive post-translation phosphorylation on serine and tyrosine residues was demonstrated by Western blot in the presence and in the absence of phosphatase inhibitors using specific antibodies. Immunoprecipitated CReMM disclosed a DNA-dependent ATPase activity quantified by colorimetric assay. Electrophoresis mobility-shift assay (EMSA) validated that CReMM binds to A/T-rich DNA. CReMM is expressed in mesenchymal progenitors, as shown in vitro and in vivo. CReMM protein structural motifs and proven biochemical activities highlight its role in chromatin remodeling. Further delineation of the function of this protein will provide information about its dynamics in transcriptional regulation of mesenchymal cells.


Context: Only a minority of the genes, identified in the Caenorhabditis elegans genome sequence data by computer analysis, have been characterized experimentally. We attempted to determine the expression patterns for a random sample of the annotated genes using reporter gene fusions. A low success rate was obtained for evolutionarily recently duplicated genes. Analysis of the data suggests that this is not due to conditional or low-level expression. The remaining explanation is that most of the annotated genes in the recently duplicated category are pseudogenes, a proportion corresponding to 20% of all of the annotated C. elegans genes. Further support for this surprisingly high figure was sought by comparing sequences for families of recently duplicated C. elegans genes. Although only a preliminary analysis, clear evidence for a gene having been recently inactivated by genetic drift was found for many genes in the recently duplicated category. At least 4% of the annotated C. elegans genes can be recognized as pseudogenes simply from closer inspection of the sequence data. Lessons learned in identifying pseudogenes in C. elegans could be of value in the annotation of the genomes of other species where, although there may be fewer pseudogenes, they may be harder to detect.


Context: The efficacy and limitations of transarterial acrylic glue embolization for the treatment of intracranial dural arteriovenous fistulas (DAVFs) were investigated. Thirty-four DAVFs treated by transarterial embolization using n-butyl cyanoacrylate were retrospectively reviewed. The locations of DAVFs were the transverse-sigmoid sinus in 11, tentorium in 10, cranial vault in 9, and superior sagittal sinus, jugular bulb, foramen magnum, and middle cranial fossa in 1 each. Borden classification was type I in 7, type II in 3, and type III in 24. Eight patients had undergone prior transvenous coil embolization. Complete obliteration rate was 56% immediately after embolization, 71% at follow-up angiography, and 85% after additional treatments (1 transvenous embolization and 4 direct surgery). Complications occurred in three patients, consisting of asymptomatic vessel perforations during cannulation in two patients and leakage of contrast medium resulting in medullary infarction in one patient. Transarterial glue embolization is highly effective for Borden type III DAVF with direct cortical venous drainage, but has limitations for Borden type I and II DAVFs in which the affected sinus is part of the normal venous circulation. Onyx is a new liquid embolic material and is becoming the treatment of choice for DAVF. The benefits of glue embolization compared to Onyx embolization are high thrombogenicity, and relatively low risks of cranial nerve palsies and of excessive migration into the draining veins of high flow fistula. Transarterial glue embolization continues to be useful for selected patients, and complete cure can be expected in most patients with fewer complications if combined with transvenous embolization or direct surgery.