Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

Abstract: We have systematically made a set of precisely defined, single‐gene deletions of all nonessential genes in Escherichia coli K‐12. Open‐reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one‐step method for inactivation of chromosomal genes and primers designed to create in‐frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high‐throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E . coli K‐12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist‐nara.ac.jp/).


Validated RealTime reverse transcriptase PCR methods for the diagnosis and pathotyping of Eurasian H7 avian influenza viruses

Abstract:
BACKGROUND Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases.
 OBJECTIVES Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping.
 METHODS The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR.
 RESULTS Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested.
 CONCLUSIONS The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.


Interleukin-6 receptor pathways in coronary heart disease: a collaborative meta-analysis of 82 studies

Abstract:
BACKGROUND Persistent inflammation has been proposed to contribute to various stages in the pathogenesis of cardiovascular disease. Interleukin-6 receptor (IL6R) signalling propagates downstream inflammation cascades. To assess whether this pathway is causally relevant to coronary heart disease, we studied a functional genetic variant known to affect IL6R signalling.
 METHODS In a collaborative meta-analysis, we studied Asp358Ala (rs2228145) in IL6R in relation to a panel of conventional risk factors and inflammation biomarkers in 125,222 participants. We also compared the frequency of Asp358Ala in 51,441 patients with coronary heart disease and in 136,226 controls. To gain insight into possible mechanisms, we assessed Asp358Ala in relation to localised gene expression and to postlipopolysaccharide stimulation of interleukin 6.
 FINDINGS The minor allele frequency of Asp358Ala was 39%. Asp358Ala was not associated with lipid concentrations, blood pressure, adiposity, dysglycaemia, or smoking (p value for association per minor allele ≥0·04 for each). By contrast, for every copy of 358Ala inherited, mean concentration of IL6R increased by 34·3% (95% CI 30·4-38·2) and of interleukin 6 by 14·6% (10·7-18·4), and mean concentration of C-reactive protein was reduced by 7·5% (5·9-9·1) and of fibrinogen by 1·0% (0·7-1·3). For every copy of 358Ala inherited, risk of coronary heart disease was reduced by 3·4% (1·8-5·0). Asp358Ala was not related to IL6R mRNA levels or interleukin-6 production in monocytes.
 INTERPRETATION Large-scale human genetic and biomarker data are consistent with a causal association between IL6R-related pathways and coronary heart disease.
 FUNDING British Heart Foundation; UK Medical Research Council; UK National Institute of Health Research, Cambridge Biomedical Research Centre; BUPA Foundation.


Quantitative analysis of tumor-derived methylated p16INK4a sequences in plasma, serum, and blood cells of hepatocellular carcinoma patients.

Abstract:
PURPOSE AND EXPERIMENTAL DESIGN Using real-time quantitative methylation-specific PCR (RTQ-MSP), we quantified methylated p16INK4a sequences and determined the fractional concentrations of circulating tumor DNA in plasma, serum, and peripheral blood cells collected preoperatively, intraoperatively, and postoperatively from 49 patients with hepatocellular carcinoma (HCC).
 RESULTS RTQ-MSP was sufficiently sensitive to detect down to 10 genome-equivalents of methylated p16INK4a sequences. Quantitative MSP data were expressed in terms of the methylation index, which was the percentage of bisulfite converted unmethylated and methylated p16INK4a sequences that consisted of methylated p16INK4a sequences. Quantities of methylated p16INK4a sequences were detected in peripheral circulation of 80% (23 of 29) of HCC patients. No significant difference was seen in the detectability and concentrations of methylated p16INK4a sequences (range: 10-4046 genome-equivalents/ml) between preoperative plasma and serum samples from HCC patients. Preoperatively, the p16INK4a methylation indices ranged from 0.2 to 100% and from 0.012 to 0.075% in the patients' plasma and buffy coat samples, respectively. After surgical resection, the median p16INK4a methylation indices in plasma and buffy coat concordantly decreased 12- and 15-fold, respectively. These results demonstrated the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring HCC after treatment. Furthermore, none of the intraoperative plasma samples and only two of the intraoperative buffy coat samples were p16INK4a methylation positive.
 CONCLUSIONS Quantification of epigenetic changes in peripheral blood by RTQ-MSP is useful for the detection and monitoring of HCC.


4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors

Abstract: Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.


Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

Abstract: All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.


Long-term financing needs for HIV control in sub-Saharan Africa in 2015-2050: a modelling study.

Abstract:
OBJECTIVES To estimate the present value of current and future funding needed for HIV treatment and prevention in 9 sub-Saharan African (SSA) countries that account for 70% of HIV burden in Africa under different scenarios of intervention scale-up. To analyse the gaps between current expenditures and funding obligation, and discuss the policy implications of future financing needs.
 DESIGN We used the Goals module from Spectrum, and applied the most up-to-date cost and coverage data to provide a range of estimates for future financing obligations. The four different scale-up scenarios vary by treatment initiation threshold and service coverage level. We compared the model projections to current domestic and international financial sources available in selected SSA countries.
 RESULTS In the 9 SSA countries, the estimated resources required for HIV prevention and treatment in 2015-2050 range from US$98 billion to maintain current coverage levels for treatment and prevention with eligibility for treatment initiation at CD4 count of <500/mm(3) to US$261 billion if treatment were to be extended to all HIV-positive individuals and prevention scaled up. With the addition of new funding obligations for HIV--which arise implicitly through commitment to achieve higher than current treatment coverage levels--overall financial obligations (sum of debt levels and the present value of the stock of future HIV funding obligations) would rise substantially.
 CONCLUSIONS Investing upfront in scale-up of HIV services to achieve high coverage levels will reduce HIV incidence, prevention and future treatment expenditures by realising long-term preventive effects of ART to reduce HIV transmission. Future obligations are too substantial for most SSA countries to be met from domestic sources alone. New sources of funding, in addition to domestic sources, include innovative financing. Debt sustainability for sustained HIV response is an urgent imperative for affected countries and donors.


Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Abstract: Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.


The pervasiveness and plasticity of circadian oscillations: the coupled circadian-oscillators framework.

Abstract:
MOTIVATION Circadian oscillations have been observed in animals, plants, fungi and cyanobacteria and play a fundamental role in coordinating the homeostasis and behavior of biological systems. Genetically encoded molecular clocks found in nearly every cell, based on negative transcription/translation feedback loops and involving only a dozen genes, play a central role in maintaining these oscillations. However, high-throughput gene expression experiments reveal that in a typical tissue, a much larger fraction ([Formula: see text]) of all transcripts oscillate with the day-night cycle and the oscillating species vary with tissue type suggesting that perhaps a much larger fraction of all transcripts, and perhaps also other molecular species, may bear the potential for circadian oscillations.
 RESULTS To better quantify the pervasiveness and plasticity of circadian oscillations, we conduct the first large-scale analysis aggregating the results of 18 circadian transcriptomic studies and 10 circadian metabolomic studies conducted in mice using different tissues and under different conditions. We find that over half of protein coding genes in the cell can produce transcripts that are circadian in at least one set of conditions and similarly for measured metabolites. Genetic or environmental perturbations can disrupt existing oscillations by changing their amplitudes and phases, suppressing them or giving rise to novel circadian oscillations. The oscillating species and their oscillations provide a characteristic signature of the physiological state of the corresponding cell/tissue. Molecular networks comprise many oscillator loops that have been sculpted by evolution over two trillion day-night cycles to have intrinsic circadian frequency. These oscillating loops are coupled by shared nodes in a large network of coupled circadian oscillators where the clock genes form a major hub. Cells can program and re-program their circadian repertoire through epigenetic and other mechanisms.    AVAILABILITY AND IMPLEMENTATION High-resolution and tissue/condition specific circadian data and networks available at http://circadiomics.igb.uci.edu.    CONTACT pfbaldi@ics.uci.edu   SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.


AMPA receptor ligand binding domain mobility revealed by functional cross linking.

Abstract: Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.


Ncf1 (p47phox) Is Essential for Direct Regulatory T Cell Mediated Suppression of CD4+ Effector T Cells

Abstract:
BACKGROUND Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs.
 METHODOLOGY/PRINCIPAL FINDINGS Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff.
 CONCLUSIONS/SIGNIFICANCE We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.


Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands.

Abstract: Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.


Expression profiles during dedifferentiation in newt lens regeneration revealed by expressed sequence tags

Abstract:
PURPOSE The adult newt can regenerate lens from pigmented epithelial cells (PECs) of the dorsal iris via dedifferentiation. The purpose of this research is to obtain sequence resources for a newt lens regeneration study and to obtain insights of dedifferentiation at the molecular level.
 METHODS mRNA was purified from iris during dedifferentiation and its cDNA library was constructed. From the cDNA library 10,449 clones were sequenced and analyzed.
 RESULTS From 10,449 reads, 780 contigs and 1,666 singlets were annotated. The presence of several cancer- and apoptosis-related genes during newt dedifferentiation was revealed. Moreover, several candidate genes, which might participate in reprogramming during dedifferentiation, were also found.
 CONCLUSIONS The expression of cancer- and apoptosis-related genes could be hallmarks during dedifferentiation. The expression sequence tag (EST) resource is useful for the future study of newt dedifferentiation, and the sequence information is available in GenBank (accession numbers; FS290155-FS300559).


In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells.

Abstract: A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.


The role of the tissue omega-6/omega-3 fatty acid ratio in regulating tumor angiogenesis

Abstract: Angiogenesis is a necessary step in tumor growth and metastasis. It is well established that the metabolites of omega-6 and omega-3 fatty acids, which must be obtained through the diet and cannot be synthesized de novo in mammals, have differential effects on cellular processes. Omega-6 fatty acid (n−6 FA)-derived metabolites promote angiogenesis by increasing growth factor expression whereas omega-3 fatty acids (n−3 FA) have anti-angiogenic and antitumor properties. However, most studies thus far have failed to account for the role of the n−6 FA/n−3 FA ratio in angiogenesis and instead examined the absolute levels of n−6 and n−3 FA. This review highlights the biochemical interactions between n−6 and n−3 FA and focuses on how the n−6/n−3 FA ratio in tissues modulates tumor angiogenesis. We suggest that future work should consider the n−6/n−3 FA ratio to be a key element in experimental design and analysis. Furthermore, we recommend that clinical interventions should aim to both reduce n−6 metabolites and simultaneously increase n−3 FA intake.


Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance.

Abstract: Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (≥1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.


Noncanonical Wnt Signaling Promotes Obesity-Induced Adipose Tissue Inflammation and Metabolic Dysfunction Independent of Adipose Tissue Expansion

Abstract: Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH2-terminal kinase-dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion.


A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease

Abstract: The 5'-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5'-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5'-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the 'A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3'- and 25S 5'-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5'-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.


Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro.

Abstract: Bisphosphonates are widely used agents for the treatment of malignant bone disease. They inhibit osteoclast-mediated bone resorption and can have direct effects on cancer cells. In this study, we investigated whether the anticancer activity of the third-generation bisphosphonate zoledronic acid (ZOL) could be enhanced by combination with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). We found that ZOL and SAHA cooperated to induce cell death in the prostate cancer cell lines LNCaP and PC-3. The effect was synergistic, as evidenced by combination index isobologram analysis. ZOL and SAHA synergized to induce dissipation of the mitochondrial transmembrane potential, to activate caspase-3, and to trigger DNA fragmentation, showing that the combination of ZOL and SAHA resulted in the initiation of apoptosis. Because ZOL acts by inhibiting the mevalonate pathway, thereby preventing protein prenylation, we explored whether the mevalonate pathway was also the target of the cooperative action of ZOL and SAHA. We found that geranylgeraniol, but not farnesol, significantly reduced ZOL/SAHA-induced cell death, indicating that the synergistic action of the agents was due to the inhibition of geranylgeranylation. Consistently, a direct inhibitor of geranylgeranylation, GGTI-298, synergized with SAHA to induce cell death, whereas an inhibitor of farnesylation, FTI-277, had no effect. In addition, SAHA synergized with mevastatin, an inhibitor of the proximal enzyme in the mevalonate pathway. These in vitro findings provide a rationale for an in vivo exploration into the potential of combining SAHA and ZOL, or other inhibitors of the mevalonate pathway, as an effective strategy for anticancer therapy.


Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4.

Abstract: Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.


Comparison of Maximal Oxygen Consumption Between Black and White Prepubertal and Pubertal Children

Abstract: The purpose of this investigation was to determine whether maximal oxygen consumption (VO2max) differed between two selected groups of black and white children and whether a difference existed to determine whether it was related to hematologic profiles, body composition, and/or physical activity/inactivity level. Forty-five prepubertal and 42 pubertal, clinically normal black and white children participated. Dual-energy x-ray absorptiometry was used to determine body composition. A computed tomography scan of the abdomen was used to determine visceral adipose tissue and s.c. adipose tissue. Daily physical activity/inactivity was assessed by questionnaire. Black prepubertal and pubertal children had lower VO2max values when compared with white children (28.8 ± 7.8 versus 35.0 ± 6.5 mL · kg−1 · min−1, p < 0.01; 33.7 ± 6.4 versus 40.4 ± 10.2 mL · kg−1 · min−1, p < 0.05; respectively). Black prepubertal and pubertal children had lower Hb concentrations ([Hb]) and hematocrits than white children (prepubertal: 12.1 ± 0.5 versus 12.8 ± 0.9 g/dL, p < 0.001; 35.6 ± 1.4 versus 37.4 ± 2.3%, p < 0.01, respectively; pubertal: 13.0 ± 0.9 versus 13.6 ± 0.7 g/dL, p < 0.05; 37.7 ± 2.5 versus 39.5 ± 2.1%, p < 0.05, respectively). In conclusion, these findings indicate that black prepubertal and pubertal children had lower VO2max when compared with their white peers matched for age, pubertal stage, and body mass index. This difference in VO2max could be attributed at least in part to comparatively lower [Hb] and more sedentary lifestyle in the black children. Further investigations should study Hb flow rate (a function of [Hb] × maximal cardiac output) in black and white children as it relates to VO2max.


Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway.

Abstract: CD4(+) helper T (Th) cells play a crucial role in the delicate balance between host defense and autoimmune disease. Two important populations of helper T cells are the proinflammatory, interleukin-17 (IL-17)-producing (Th17) cells and the anti-inflammatory forkhead box P3-positive (FoxP3(+)) T regulatory (Treg) cells. Here we show that all-trans retinoic acid (ATRA) and other agonists of the retinoic acid receptor alpha (RARalpha) inhibit the formation of Th17 cells and promote FoxP3 expression. Conversely, inhibition of retinoic acid signaling constrains transforming growth factor beta (TGF-beta1) induction of FoxP3. The effect of ATRA is mediated independently of IL-2, signal transducer and activator of transcription 5 (Stat5) and Stat3, representing a novel mechanism for the induction of FoxP3 in CD4 T cells. As previous studies have shown that vitamin A derivatives are protective in animal models of autoimmune disease, the current data suggest a previously unrecognized role for RARalpha in the regulation of CD4(+) T-cell differentiation and provide a mechanism for the anti-inflammatory effects of retinoic acid.


Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI)

Abstract: Superresolution Optical Fluctuation Imaging (SOFI) as initially demonstrated allows for a resolution enhancement in imaging by a factor of square-root of two. Here, we demonstrate how to increase the resolution of SOFI images by re-weighting the Optical Transfer Function (OTF). Furthermore, we demonstrate how cross-cumulants can be exploited to obtain a fair approximation of the underlying Point-Spread Function. We show a two-fold increase of resolution (over the diffraction limit) of near-infrared quantum dot labeled tubulin-network of 3T3 fibroblasts.


Evaluation of reagent strips in detecting asymptomatic bacteriuria in early pregnancy: prospective case series.

Abstract:
OBJECTIVE To evaluate the performance of reagent test strips in screening pregnant women for asymptomatic bacteriuria at their first visit to an antenatal clinic.
 DESIGN Prospective case series.
 SETTING Antenatal clinic of a large inner city maternity hospital.    SUBJECTS All women attending for their first antenatal clinic. Patients taking antibiotics for any reason and those with urinary tract symptoms were excluded.
 INTERVENTION A midstream urine specimen was divided; half was sent for microscopy and formal bacteriological culture and the other half was tested with a commercial reagent strip test for the presence of blood, protein, nitrite, and leucocyte esterase.
 MAIN OUTCOME MEASURES Sensitivity, specificity, and positive and negative predictive values of the reagent strips in diagnosing asymptomatic bacteriuria (defined as 10(5) colony forming units/ml urine).
 RESULTS Sensitivity was low, with a maximum of 33% when all four tests were used in combination. Specificity was high, with typical values of 99% or more. Positive predictive value reached a maximum of 69% and negative predictive value was typically 95% or more.
 CONCLUSION Urine reagent strips are not sufficiently sensitive to be of use in the screening for asymptomatic bacteriuria and therefore many patients would be missed. In view of the potentially serious sequelae of this condition in pregnant women we recommend that formal bacteriological investigation remain the investigation of choice in this group of patients.


How reliable are light traps in estimating biting rates of adult Anopheles gambiae s.l. (Diptera: Culicidae) in the presence of treated bed nets?

Abstract: The sampling efficiency of light trap catches relative to human bait catches in estimating biting rates of the mosquito Anopheles gambiae Giles was investigated in two types of community in southern Sierra Leone: (i) where most of the inhabitants slept under treated bed nets; and (ii) where most of the inhabitants slept without bed nets. The number of female A. gambiae mosquitoes caught in these communities by light trap was strongly correlated (r > or = 0.72) with those from corresponding human biting catches performed either on the same or adjacent nights. It was found that the relative sampling efficiency of light traps varied slightly but significantly with mosquito abundance in villages with treated bed nets, but not in those without them. Nevertheless, the relationship between relative sampling efficiency and mosquito abundance did not differ significantly between the two types of village. Overall, there was insufficient evidence to show that the presence of treated nets altered the relative efficiency of light traps and any bias was only slight, and unlikely to be of any practical importance. Hence, it was concluded that light traps can be used as a surrogate for human bait catches in estimating biting rates of A. gambiae mosquitoes in the two communities.


NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome

Abstract: Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.


Periostin Deficiency Increases Bone Damage and Impairs Injury Response to Fatigue Loading in Adult Mice

Abstract: Bone damage removal and callus formation in response to fatigue loading are essential to prevent fractures. Periostin (Postn) is a matricellular protein that mediates adaptive response of cortical bone to loading. Whether and how periostin influences damage and the injury response to fatigue remains unknown. We investigated the skeletal response of Postn(-/-) and Postn(+/+) mice after fatigue stimulus by axial compression of their tibia. In Postn(+/+) mice, cracks number and surface (CsNb, CsS) increased 1h after fatigue, with a decrease in strength compared to non-fatigued tibia. At 15 days, CsNb had started to decline, while CtTV and CtBV increased in fatigued vs non-fatigued tibia, reflecting a woven bone response that was present in 75% of the fatigued bones. Cortical porosity and remodelling also prominently increased in the fatigued tibia of Postn(+/+) mice. At 30 days, paralleling a continuous removal of cortical damage, strength of the fatigued tibia was similar to the non-fatigue tibia. In Postn(-/-) mice, cracks were detectable even in the absence of fatigue, while the amount of collagen crosslinks and tissue hardness was decreased compared to Postn(+/+). Fatigue significantly increased CsNb and CsS in Postn(-/-), but was not associated with changes in CtTV and CtBV, as only 16% of the fatigued bones formed some woven bone. Cortical porosity and remodelling did not increase either after fatigue in Postn(-/-), and the level of damage remained high even after 30 days. As a result, strength remained compromised in Postn(-/-) mice. Contrary to Postn(+/+), which osteocytic lacunae showed a change in the degree of anisotropy (DA) after fatigue, Postn(-/-) showed no DA change. Hence periostin appears to influence bone materials properties, damage accumulation and repair, including local modeling/remodeling processes in response to fatigue. These observations suggest that the level of periostin expression could influence the propensity to fatigue fractures.


Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling.

Abstract: The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation.


Genomic organisation and transcription characterisation of the gene encoding Leishmania (Leishmania) amazonensis arginase and its protein structure prediction.

Abstract: The genomic organisation of the gene encoding Leishmania (Leishmania) amazonensis arginase as well as its flanking regions were characterised. The size of the transcribed RNA was determined, allowing us to map the genomic sites signalling for RNA trans-splicing and putative polyadenylation regions. The general organisation was compared with genes encoding other proteins already described in organisms of the Trypanosomatid family. The complete nucleotide sequence of the arginase open reading frame was obtained and the three-dimensional structure of the enzyme was inferred by a computational analysis of the deduced amino acid sequence, based on the established crystal structure described for Rattus norvergicus arginase. The human liver arginase sequence was analysed in the same way and the comparison of the presumed structure of both the Leishmania and human enzymes identified some differences that may be exploited in chemotherapeutic studies.


Redefining the Genetic Hierarchies Controlling Skeletal Myogenesis: Pax-3 and Myf-5 Act Upstream of MyoD

Abstract: We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.


Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

Abstract: How oncogenic signalling coordinates glycolysis and anabolic biosynthesis in cancer cells remains unclear. We recently reported that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate and product 2-phosphoglycerate. Here we report a novel mechanism in which Y26 phosphorylation enhances PGAM1 activation through release of inhibitory E19 that blocks the active site, stabilising cofactor 2,3-bisphosphoglycerate binding and H11 phosphorylation. We also report the crystal structure of H11-phosphorylated PGAM1 and find that phospho-H11 activates PGAM1 at least in part by promoting substrate 3-phosphoglycerate binding. Moreover, Y26 phosphorylation of PGAM1 is common in human cancer cells and contributes to regulation of 3-phosphoglycerate and 2-phosphoglycerate levels, promoting cancer cell proliferation and tumour growth. As PGAM1 is a negative transcriptional target of TP53, and is therefore commonly upregulated in human cancers, these findings suggest that Y26 phosphorylation represents an additional acute mechanism underlying phosphoglycerate mutase 1 upregulation.


Spatial and Temporal Clustering of Dengue Virus Transmission in Thai Villages

Abstract:
BACKGROUND Transmission of dengue viruses (DENV), the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted.
 METHODS AND FINDINGS Cluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters) and without (negative clusters) acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely (1) define the spatial and temporal dimensions of DENV transmission, (2) correlate these factors with variation in DENV transmission, and (3) determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees) occurred in positive clusters (p < 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1-19.8 per 100]. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8%) dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between positive and negative clusters were greater availability of piped water in negative clusters (p < 0.01) and greater number of Ae. aegypti pupae per person in positive clusters (p = 0.04). During primarily DENV-4 transmission seasons, the ratio of inapparent to symptomatic infections was nearly 1:1 among child enrollees. Study limitations included inability to sample all children and mosquitoes within each cluster and our reliance on serologic rather than virologic evidence of interval infections in enrollees given restrictions on the frequency of blood collections in children.
 CONCLUSIONS Our data reveal the remarkably focal nature of DENV transmission within a hyperendemic rural area of Thailand. These data suggest that active school-based dengue case detection prompting local spraying could contain recent virus introductions and reduce the longitudinal risk of virus spread within rural areas. Our results should prompt future cluster studies to explore how host immune and behavioral aspects may impact DENV transmission and prevention strategies. Cluster methodology could serve as a useful research tool for investigation of other temporally and spatially clustered infectious diseases.


The Histone H3 Lysine-27 Demethylase Jmjd3 Links Inflammation to Inhibition of Polycomb-Mediated Gene Silencing

Abstract: Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.


Mental Health Conditions Among Patients Seeking and Undergoing Bariatric Surgery: A Meta-analysis.

Abstract:
IMPORTANCE Bariatric surgery is associated with sustained weight loss and improved physical health status for severely obese individuals. Mental health conditions may be common among patients seeking bariatric surgery; however, the prevalence of these conditions and whether they are associated with postoperative outcomes remains unknown.
 OBJECTIVE To determine the prevalence of mental health conditions among bariatric surgery candidates and recipients, to evaluate the association between preoperative mental health conditions and health outcomes following bariatric surgery, and to evaluate the association between surgery and the clinical course of mental health conditions.
 DATA SOURCES We searched PubMed, MEDLINE on OVID, and PsycINFO for studies published between January 1988 and November 2015. Study quality was assessed using an adapted tool for risk of bias; quality of evidence was rated based on GRADE (Grading of Recommendations Assessment, Development and Evaluation) criteria.
 FINDINGS We identified 68 publications meeting inclusion criteria: 59 reporting the prevalence of preoperative mental health conditions (65,363 patients) and 27 reporting associations between preoperative mental health conditions and postoperative outcomes (50,182 patients). Among patients seeking and undergoing bariatric surgery, the most common mental health conditions, based on random-effects estimates of prevalence, were depression (19% [95% CI, 14%-25%]) and binge eating disorder (17% [95% CI, 13%-21%]). There was conflicting evidence regarding the association between preoperative mental health conditions and postoperative weight loss. Neither depression nor binge eating disorder was consistently associated with differences in weight outcomes. Bariatric surgery was, however, consistently associated with postoperative decreases in the prevalence of depression (7 studies; 8%-74% decrease) and the severity of depressive symptoms (6 studies; 40%-70% decrease).
 CONCLUSIONS AND RELEVANCE Mental health conditions are common among bariatric surgery patients-in particular, depression and binge eating disorder. There is inconsistent evidence regarding the association between preoperative mental health conditions and postoperative weight loss. Moderate-quality evidence supports an association between bariatric surgery and lower rates of depression postoperatively.


Contractile forces sustain and polarize hematopoiesis from stem and progenitor cells.

Abstract: Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.


Functional insights into the role of nuclear-retained long noncoding RNAs in gene expression control in mammalian cells

Abstract: The mammalian genome harbors thousands of long noncoding RNA (lncRNA) genes. Recent studies have indicated the involvement of several of these lncRNAs in the regulation of gene expression. lncRNAs play crucial roles in various biological processes ranging from epigenetic gene regulation, transcriptional control, to post-transcriptional regulation. lncRNAs are localized in various subcellular compartments, and major proportion of these are retained in the cell nucleus and could be broadly classified as nuclear-retained lncRNAs (nrRNAs). Based on the identified functions, members of the nrRNAs execute diverse roles, including providing architectural support to the hierarchical subnuclear organization and influencing the recruitment of chromatin modifier factors to specific chromatin sites. In this review, we will summarize the recently described roles of mammalian nrRNAs in controlling gene expression by influencing chromatin organization, transcription, pre-mRNA processing, nuclear organization, and their involvement in disease.


TGF-β and Insulin Signaling Regulate Reproductive Aging via Oocyte and Germline Quality Maintenance

Abstract: Reproductive cessation is perhaps the earliest aging phenotype that humans experience. Similarly, reproduction of Caenorhabditis elegans ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here, we show that TGF-β Sma/Mab and Insulin/IGF-1 signaling regulate C. elegans reproductive aging by modulating multiple aspects of the reproductive process, including embryo integrity, oocyte fertilizability, chromosome segregation fidelity, DNA damage resistance, and oocyte and germline morphology. TGF-β activity regulates reproductive span and germline/oocyte quality noncell-autonomously and is temporally and transcriptionally separable from its regulation of growth. Chromosome segregation, cell cycle, and DNA damage response genes are upregulated in TGF-β mutant oocytes, decline in aged mammalian oocytes, and are critical for oocyte quality maintenance. Our data suggest that C. elegans and humans share many aspects of reproductive aging, including the correlation between reproductive aging and declining oocyte quality and mechanisms determining oocyte quality.


NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

Abstract: Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.


Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

Abstract: The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.


Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome

Abstract: The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.


Fibrillar Collagen Inhibits Arterial Smooth Muscle Proliferation through Regulation of Cdk2 Inhibitors

Abstract: Arterial smooth muscle cells (SMCs) are arrested in the G1 phase of the cell cycle on polymerized type I collagen fibrils, while monomer collagen supports SMC proliferation. Cyclin E-associated kinase and cyclin-dependent kinase 2 (cdk2) phosphorylation are inhibited on polymerized collagen, and levels of the cdk2 inhibitors p27Kip1 and p21Cip1/Waf1 are increased compared with SMCs on monomer collagen. p27Kip1 associates with the cyclin E-cdk2-p21Cip1/Waf1 complex in SMCs on polymerized collagen. Monovalent blocking antibodies to alpha2 integrins, integrins that mediate adhesion to both forms of collagen, mimic these effects on monomer collagen. Furthermore, polymerized collagen rapidly suppresses p70 S6 kinase, a possible regulator of p27Kip1. Thus, fibrillar collagen specifically regulates early integrin signaling that may lead to up-regulation of cdk2 inhibitors and inhibition of SMC proliferation.


Direct Upregulation of STAT3 by MicroRNA-551b-3p Deregulates Growth and Metastasis of Ovarian Cancer.

Abstract: 3q26.2 amplification in high-grade serous ovarian cancer leads to increased expression of mature microRNA miR551b-3p, which is associated with poor clinical outcome. Importantly, miR551b-3p contributes to resistance to apoptosis and increased survival and proliferation of cancer cells in vitro and in vivo. miR551b-3p upregulates STAT3 protein levels, and STAT3 is required for the effects of miR551b-3p on cell proliferation. Rather than decreasing levels of target mRNA as expected, we demonstrate that miR551b-3p binds a complementary sequence on the STAT3 promoter, recruiting RNA polymerase II and the TWIST1 transcription factor to activate STAT3 transcription, and thus directly upregulates STAT3 expression. Furthermore, anti-miR551b reduced STAT3 expression in ovarian cancer cells in vitro and in vivo and reduced ovarian cancer growth in vivo. Together, our data demonstrate a role for miR551b-3p in transcriptional activation. Thus, miR551b-3p represents a promising candidate biomarker and therapeutic target in ovarian cancer.


Mental Health Support Provided Throughout the Bariatric Surgery Clinical Pathway in French Specialized Care Centers for Obesity

Abstract: Pre-operative psychological assessment is recommended by international guidelines for bariatric surgery candidates. Thereby, service teams caring for bariatric patients should include at least one mental health provider (e.g., a psychologist or psychiatrist). The objective of this study was to evaluate the psychology and psychiatry resources and practices in the 37 specialized obesity centers (CSOs) created by the French Ministry of Health. CSO coordinators were contacted by e-mail to collect general information on the centers (e.g., number of bariatric operations). Secondly, psychologists and psychiatrists of each center completed an anonymous questionnaire assessing their professional practices and their organization of care pathways. The vast majority of CSO coordinators (81%, n = 26/32) answered our survey. These results show significant differences and shortages in terms of the psychology/psychiatry resources available. Most of the psychologists (n = 26/31) and psychiatrists (n = 10/10) stated that they systematically meet new patients only before surgery (56%) or both before and after the operation (30%); however, some psychologists and psychiatrists (14%) do not systematically meet all the patients (before and/or after surgery). Nevertheless, all the professionals provide psychology assessments, and about 75% of them offer a psychological follow-up, indicating a similarity regarding the practices of psychologists and psychiatrists. Our results highlight the place of psychological/psychiatric evaluations in French CSOs and emphasize the absence of mental health providers in several of these services. Post-operative psychological follow-up is not usually provided. It would be appropriate to create clear recommendations for post-operative psychological or psychiatric long-term follow-up.


Breaking the code of DNA binding specificity of TAL-type III effectors.

Abstract: The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.


Good and bad consequences of altered fatty acid metabolism in heart failure: evidence from mouse models.

Abstract: The shift in substrate preference away from fatty acid oxidation (FAO) towards increased glucose utilization in heart failure has long been interpreted as an oxygen-sparing mechanism. Inhibition of FAO has therefore evolved as an accepted approach to treat heart failure. However, recent data indicate that increased reliance on glucose might be detrimental rather than beneficial for the failing heart. This review discusses new insights into metabolic adaptations in heart failure. A particular focus lies on data obtained from mouse models with modulations of cardiac FA metabolism at different levels of the FA metabolic pathway and how these differently affect cardiac function. Based on studies in which these mouse models were exposed to ischaemic and non-ischaemic heart failure, we discuss whether and when modulations in FA metabolism are protective against heart failure.


Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells

Abstract: Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP+ cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient–X-linked, mouse muscular dystrophy (scid–mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.


UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis

Abstract: Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.


In-hospital switching of oral P2Y12 inhibitor treatment in patients with acute coronary syndrome undergoing percutaneous coronary intervention: prevalence, predictors and short-term outcome.

Abstract:
BACKGROUND P2Y12 inhibitor switching has appeared in clinical practice as a consequence of prasugrel and ticagrelor availability, apart from clopidogrel, for use in patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI).
 METHODS In the context of the GReek AntiPlatelet REgistry (GRAPE) we assessed the prevalence, predictive factors and short-term outcome of in-hospital P2Y12 inhibitor switching in 1794 ACS patients undergoing PCI.
 RESULTS Switching occurred in 636 (35.5%) patients of which in the form of clopidogrel to a novel agent, novel agent to clopidogrel and between prasugrel and ticagrelor in 574 (90.4%), 34 (5.3%) and 27 (4.3%) patients, respectively. Presentation to non PCI-capable hospital, bivalirudin use, age ≥75 years (inverse predictor), and regional trends emerged as predictive factors of switching to a novel agent. At combined in-hospital and one-month follow-up, propensity matched pairs analysis showed no differences in major adverse cardiovascular (MACE) or bleeding events between switching from clopidogrel to a novel agent vs novel agent constant administration. More Bleeding Academic Research Consortium type 1, type 2 and any type events and fewer MACE were seen when switching from clopidogrel to a novel agent vs only clopidogrel administration (23.7%, 3.8%, 30.6%, 1.2% vs 8.9%, 1.2%, 12.0%, 3.8% with P < .001, P = .03, P < .001 and P = .03 respectively).
 CONCLUSIONS In a real-life experience with contemporary antiplatelet treatment in ACS patients undergoing PCI, in-hospital switching represents common clinical practice. Clinical factors and regional practice differences seem to affect this strategy's choice, while switching to a novel agent may be associated with higher risk of bleeding.


Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding.

Abstract: The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.


Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys).

Abstract: Sera from 526 Old-World monkeys and apes, representing 50 species and 20 genera and living in US zoos and vivaria, were screened for antibodies to HTLV-I, HTLV-III/LAV, and simian-AIDS retrovirus, type I (SRV-I). Sera were screened initially by ELISA, and ELISA-positive sera, as well as ELISA-negative sera from cage contacts, were further tested by Western blotting. A large number of false-positive and a small number of false-negative ELISA sera were identified. Although most true positive reactions were directed to a single retrovirus, a number of individuals from 4 species were positive for more than one retrovirus. Specific seroreactivity to HTLV-I was found in 39/526 (7%) animals of 15 species. True positive reactions to SRV-I were found in 21/516 (4%) animals, including talapoins and 2 species of macaques. Specific serologic reactions to HTLV-III/LAV were detected in 23/526 (4%) monkeys. Many of the HTLV-III/LAV seropositive animals were from one mixed-species zoo exhibit, containing sooty mangabeys, mandrills, Kolb's guenons, and talapoins. A type D virus was isolated from the blood of 3/10 SRV-I antibody-positive Tonkeana macaques, but from none of 11 seropositive talapoins. A lentivirus was isolated from the blood of 4/7 HTLV-III/LAV seropositive sooty mangabeys, but not from seropositive talapoins in the same exhibit or from 2 seropositive colobus from another zoo. The sooty mangabey lentivirus produced generalized lymphadenopathy, leukopenia, and decreased levels of T4 lymphocytes in 2 experimentally infected rhesus macaques.


p16INK4A is a robust in vivo biomarker of cellular aging in human skin.

Abstract: The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.


hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity.

Abstract: Prim-pol is a recently identified DNA primase-polymerase belonging to the archaeao-eukaryotic primase (AEP) superfamily. Here, we characterize a previously unrecognized prim-pol in human cells, which we designate hPrimpol1 (human primase-polymerase 1). hPrimpol1 possesses primase and DNA polymerase activities in vitro, interacts directly with RPA1 and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in the restart of stalled replication forks. Both RPA1 binding and the primase activity of hPrimpol1 are required for its cellular function during DNA replication. Our results indicate that hPrimpol1 is a novel factor involved in the response to DNA replication stress.


The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma.

Abstract:
BACKGROUND Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the serum and tissue expression of miR 21 and miR 199-a to be applied as early detectors for HCC.
 METHODS A total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-21 and miR-199-a) were detected by real time quantitative reverse-transcriptase PCR (RT-qPCR) in sera and tissues of chronic hepatitis and HCC patients. Besides, miR-21 and miR-199-a levels in relation to clinical and pathological factors were explored.
 RESULTS We found that the expression of serum miR-21 was distinctly increased in HCC compared with chronic hepatitis (P<0.001). miR 199-a was distinctly decreased in HCC compared with chronic hepatitis (P<0.001). In addition, median of miR 21 was increased in malignant when compared to adjacent non-malignant tissues without significant differences (P=0.191) while miR 199-a was significantly decreased in malignant when compared to adjacent nonmalignant tissues (P<0.001). ROC analysis showed that miR-21 and miR-199-a might be potential biomarkers for HCC.
 CONCLUSIONS In conclusion, the expression of miR-21 was significantly up-regulated and miR-199-a was significantly down regulated in serum of patients with HCC. Due to their reasonable sensitivity and specificity for disease progression, miR-21 and miR-199-a could be used as potential circulating biomarkers for HCC.


Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming.

Abstract: Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.


Value of whole brain re-irradiation for brain metastases--single centre experience.

Abstract:
AIMS There is controversy in published studies regarding the role of repeat whole brain radiation (WBRT) for previously irradiated brain metastases. The aim of our retrospective study was to document the practice at Princess Margaret Hospital with respect to the re-irradiation of patients with progressive or recurrent brain metastatic disease after initial WBRT.    MATERIALS AND METHODS A comprehensive computerised database was used to identify patients treated for brain metastases with more than one course of WBRT between 1997 and 2003. Seventy-two patients were treated with WBRT for brain metastases and retreated with WBRT at a later date. The records of these patients were reviewed.
 RESULTS The median age was 56.5 years. The most common primary sites were lung (51 patients) and breast (17 patients). The most frequent dose used for the initial radiotherapy was 20 Gy/5 fractions (62 patients). The most common doses of re-irradiation were 25 Gy/10 fractions (22 patients), 20 Gy/10 fractions (12 patients), 15 Gy/5 fractions (11 patients) and 20 Gy/8 fractions (10 patients). Thirty-one per cent of patients experienced a partial clinical response after re-irradiation, as judged by follow-up clinical notes; 27% remained stable; 32% deteriorated after re-irradiation. Patients who had Eastern Cooperative Oncology Group performance status 0-1 at the time of retreatment lived longer. In responders, the mean duration of response was 5.1 months. The median survival after re-irradiation was 4.1 months. One patient was reported as having memory impairment and pituitary insufficiency after 5 months of progression-free survival.
 CONCLUSION Repeat radiotherapy may be a useful treatment in carefully selected patients. With increased survival and better systemic options for patients with metastatic disease, more patients may be candidates for consideration of repeat WBRT for recurrent brain metastases, but prospective studies are needed to more clearly document their outcomes.


IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes.

Abstract: Inflammation occurs in adipose tissue in obesity. We have examined whether IL-33, a recently identified IL-1 gene family member, and its associated receptors are expressed in human adipocytes. IL-33, IL-1RL1 and IL-1RAP gene expression was observed in human visceral white fat, in preadipocytes and in adipocytes (SGBS cells). Treatment with TNFalpha for 24h induced a 6-fold increase in IL-33 mRNA level in preadipocytes and adipocytes. Time-course studies with adipocytes showed that the increase in IL-33 mRNA with TNFalpha was maximal (>55-fold) at 12h. This response was markedly different to IL-1beta (peak mRNA increase at 2h; 5.4-fold) and 1L-18 (peak mRNA increase at 6h; >1500-fold). Exposure of adipocytes to hypoxia (1% O(2), 24h) did not alter IL-33 mRNA level; in preadipocytes, however, there was a 3-fold increase. Human adipocytes and preadipocytes express IL-33, but the various IL-1 family members exhibit major differences in responsiveness to TNFalpha.


Extending healthy life span--from yeast to humans.

Abstract: When the food intake of organisms such as yeast and rodents is reduced (dietary restriction), they live longer than organisms fed a normal diet. A similar effect is seen when the activity of nutrient-sensing pathways is reduced by mutations or chemical inhibitors. In rodents, both dietary restriction and decreased nutrient-sensing pathway activity can lower the incidence of age-related loss of function and disease, including tumors and neurodegeneration. Dietary restriction also increases life span and protects against diabetes, cancer, and cardiovascular disease in rhesus monkeys, and in humans it causes changes that protect against these age-related pathologies. Tumors and diabetes are also uncommon in humans with mutations in the growth hormone receptor, and natural genetic variants in nutrient-sensing pathways are associated with increased human life span. Dietary restriction and reduced activity of nutrient-sensing pathways may thus slow aging by similar mechanisms, which have been conserved during evolution. We discuss these findings and their potential application to prevention of age-related disease and promotion of healthy aging in humans, and the challenge of possible negative side effects.


Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.

Abstract: The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.


N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements.

Abstract: Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. We show here, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs.


Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.

Abstract: We report an inborn error of the tricarboxylic acid cycle, fumarase deficiency, in two siblings born to first cousin parents. They presented with progressive encephalopathy, dystonia, leucopenia, and neutropenia. Elevation of lactate in the cerebrospinal fluid and high fumarate excretion in the urine led us to investigate the activities of the respiratory chain and of the Krebs cycle, and to finally identify fumarase deficiency in these two children. The deficiency was profound and present in all tissues investigated, affecting the cytosolic and the mitochondrial fumarase isoenzymes to the same degree. Analysis of fumarase cDNA demonstrated that both patients were homozygous for a missense mutation, a G-955-->C transversion, predicting a Glu-319-->Gln substitution. This substitution occurred in a highly conserved region of the fumarase cDNA. Both parents exhibited half the expected fumarase activity in their lymphocytes and were found to be heterozygous for this substitution. The present study is to our knowledge the first molecular characterization of tricarboxylic acid deficiency, a rare inherited inborn error of metabolism in childhood.


A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia

Abstract: Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p. Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; Pcombined = 4.88 × 10−94). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10−4). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.


Uptake of rosuvastatin by isolated rat hepatocytes: comparison with pravastatin.

Abstract: 1. The liver is the target organ for the lipid-regulating effect of rosuvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and liver-selective uptake of this drug is therefore a desirable property. The uptake kinetics of rosuvastatin were investigated and compared with those of pravastatin using isolated rat hepatocytes. 2. Uptake for both drugs involved both active transport and passive diffusion processes. The Michaelis constant (K(m)) of uptake rate for rosuvastatin (9.17 micro M) was approximately half that for pravastatin (16.5 micro M). However, the maximum uptake rate (V(max)) and carrier-mediated uptake clearance (V(max)/K(m)) of rosuvastatin were significantly (p < 0.01) greater than those of pravastatin, and a larger contribution of carrier-mediated uptake clearance to total uptake clearance was shown for rosuvastatin (contribution ratio 0.903 versus pravastatin 0.654). 3. Sodium and chloride ions did not play a significant role in the uptake of rosuvastatin and pravastatin, but the uptake of both drugs was inhibited both by depletion of cellular ATP and by organic anions such as bromosulfophthalein. 4. Rosuvastatin competitively inhibited the uptake of pravastatin, with an inhibition constant (K(i)) (2.75 micro M) relatively similar to its K(m). 5. The results suggest that an organic anion transport protein is the main mediator of the hepatic uptake of rosuvastatin and pravastatin, which occurs in an ATP-dependent manner. Our results indicated that rosuvastatin was taken up by the hepatocytes via the same transport systems as pravastatin, but with a greater affinity and efficiency than pravastatin.


The PI3K inhibitor arsenal: choose your weapon!

Abstract: Owing to its widespread activation in inflammation and cancer, a growing appreciation of the therapeutic potential of inhibitors of the phosphoinositide 3-kinase (PI3K) pathway has stimulated intense interest in compounds with suitable pharmacological profiles. These are primarily directed toward PI3K itself. However, as class I PI3Ks are also essential for a range of normal physiological processes, broad spectrum PI3K inhibition could be poorly tolerated. In recent years, patents describing a new generation of PI3K inhibitors have started to appear, with a particular focus on the development of compounds with enhanced isoform selectivity for use as anti-cancer and anti-inflammatory therapies. However, challenges remain for the efforts to pharmacologically target this enzyme family in a successful manner.


Infralimbic BDNF/TrkB enhancement of GluN2B currents facilitates extinction of a cocaine-conditioned place preference.

Abstract: Brain-derived neurotrophic factor (BDNF) regulates synaptic activity and behavioral flexibility, and reduction of BDNF strongly predicts psychiatric disorders and cognitive dysfunction. Restoration of BDNF-dependent activity could alleviate these impairments, but BDNF has limited clinical utility due to its pharmacokinetics. Here we demonstrate that activation of a primary BDNF target, the tropomyosin-related kinase B (TrkB) receptor, enhances the amplitude and prolongs the decay kinetics of N-methyl-d-aspartate receptor (NMDAR) currents in male rat infralimbic prefrontal pyramidal neurons. Moreover, these effects were prevented and reversed by blockade of NMDARs containing the GluN2B subunit. Our results show that this signaling cascade bidirectionally regulates extinction of a cocaine-induced conditioned place preference (CPP), a task that requires behavioral flexibility. Blockade of infralimbic TrkB receptors or GluN2B-containing NMDARs disrupted consolidation of extinction of the CPP. In contrast, extinction was strengthened by potentiation of TrkB receptor activity with infralimbic infusions of BDNF or systemic injections of 7,8 dihydroxyflavone (7,8DHF), the newly synthesized TrkB receptor agonist. The 7,8DHF-induced enhancement of extinction was prevented by infralimbic infusions of a GluN2B-specific receptor antagonist, demonstrating that TrkB receptor activation enhances extinction of cocaine-CPP via GluN2B-containing NMDARs. Together, infralimbic TrkB receptor activation strengthens GluN2B-containing NMDAR currents to support extinction learning. TrkB receptor agonists would therefore be useful as pharmacological adjuncts for extinction-based therapies for treatment of psychiatric disorders associated with reduced BDNF activity.


Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge.

Abstract: Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.


Balancing co-stimulation and inhibition with BTLA and HVEM

Abstract: The interaction between B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor whose extracellular domain belongs to the immunoglobulin superfamily, and herpesvirus-entry mediator (HVEM), a co-stimulatory tumour-necrosis factor receptor, is unique in that it is the only receptor–ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and what downstream signalling events might occur as a result of receptor ligation. As we discuss, recent studies show that engagement of HVEM with its endogenous ligand (LIGHT) from the tumour-necrosis factor family induces a powerful immune response, whereas HVEM interactions with BTLA negatively regulate T-cell responses.


Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.

Abstract: Lin28a/b are RNA-binding proteins that influence stem cell maintenance, metabolism, and oncogenesis. Poorly differentiated, aggressive cancers often overexpress Lin28, but its role in tumor initiation or maintenance has not been definitively addressed. We report that LIN28B overexpression is sufficient to initiate hepatoblastoma and hepatocellular carcinoma in murine models. We also detected Lin28b overexpression in MYC-driven hepatoblastomas, and liver-specific deletion of Lin28a/b reduced tumor burden, extended latency, and prolonged survival. Both intravenous siRNA against Lin28b and conditional Lin28b deletion reduced tumor burden and prolonged survival. Igf2bp proteins are upregulated, and Igf2bp3 is required in the context of LIN28B overexpression to promote growth. Therefore, multiple murine models demonstrate that Lin28b is both sufficient to initiate liver cancer and necessary for its maintenance.


Nanoenzymology of the 20S proteasome: proteasomal actions are controlled by the allosteric transition.

Abstract: The proteasome is a major cytosolic proteolytic assembly, essential for the physiology of eukaryotic cells. Both the architecture and enzymatic properties of the 20S proteasome are relatively well understood. However, despite longstanding interest, the integration of structural and functional properties of the proteasome into a coherent model explaining the mechanism of its enzymatic actions has been difficult. Recently, we used tapping mode atomic force microscopy (AFM) in liquid to demonstrate that the alpha-rings of the proteasome imaged in a top-view position repeatedly switched between their open and closed conformations, apparently to control access to the central channel. Here, we show with AFM that the molecules in a side-view position acquired two stable conformations. The overall shapes of the 20S particles were classified as either barrel-like or cylinder-like. The relative abundance of the two conformers depended on the nature of their interactions with ligands. Similarly to the closed molecules in top view, the barrels predominated in control or inhibited molecules. The cylinders and open molecules prevailed when the proteasome was observed in the presence of peptide substrates. Based on these data, we developed the two-state model of allosteric transitions to explain the dynamics of proteasomal structure. This model helps to better understand the observed properties of the 20S molecule, and sets foundations for further studies of the structural dynamics of the proteasome.