title: A case-mix classification system for explaining healthcare costs using administrative data in Italy.

text: BACKGROUND The Italian National Health Service (NHS) provides universal coverage to all citizens, granting primary and hospital care with a copayment system for outpatient and drug services. Financing of Local Health Trusts (LHTs) is based on a capitation system adjusted only for age, gender and area of residence. We applied a risk-adjustment system (Johns Hopkins Adjusted Clinical Groups System, ACG® System) in order to explain health care costs using routinely collected administrative data in the Veneto Region (North-eastern Italy). METHODS All residents in the Veneto Region were included in the study. The ACG system was applied to classify the regional population based on the following information sources for the year 2015: Hospital Discharges, Emergency Room visits, Chronic disease registry for copayment exemptions, ambulatory visits, medications, the Home care database, and drug prescriptions. Simple linear regressions were used to contrast an age-gender model to models incorporating more comprehensive risk measures aimed at predicting health care costs. RESULTS A simple age-gender model explained only 8% of the variance of 2015 total costs. Adding diagnoses-related variables provided a 23% increase, while pharmacy based variables provided an additional 17% increase in explained variance. The adjusted R-squared of the comprehensive model was 6 times that of the simple age-gender model. CONCLUSIONS ACG System provides substantial improvement in predicting health care costs when compared to simple age-gender adjustments. Aging itself is not the main determinant of the increase of health care costs, which is better explained by the accumulation of chronic conditions and the resulting multimorbidity.


title: EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes.

text: BACKGROUND & AIMS Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. METHODS Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. RESULTS EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. CONCLUSIONS The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. LAY SUMMARY In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.


title: Parathyroid Hormone Directs Bone Marrow Mesenchymal Cell Fate.

text: Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1+RANKL+ marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate.


title: Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion

text: BACKGROUND Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms. METHODS Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways. RESULTS Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect. CONCLUSIONS Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.


title: Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin.

text: Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.


title: Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia.

text: BACKGROUND Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. NEW METHOD The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. RESULTS We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. COMPARISON WITH EXISTING METHOD The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. CONCLUSION We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment.


title: Sugar-sweetened beverages and weight gain in children and adults: a systematic review and meta-analysis.

text: BACKGROUND The relation between sugar-sweetened beverages (SSBs) and body weight remains controversial. OBJECTIVE We conducted a systematic review and meta-analysis to summarize the evidence in children and adults. DESIGN We searched PubMed, EMBASE, and Cochrane databases through March 2013 for prospective cohort studies and randomized controlled trials (RCTs) that evaluated the SSB-weight relation. Separate meta-analyses were conducted in children and adults and for cohorts and RCTs by using random- and fixed-effects models. RESULTS Thirty-two original articles were included in our meta-analyses: 20 in children (15 cohort studies, n = 25,745; 5 trials, n = 2772) and 12 in adults (7 cohort studies, n = 174,252; 5 trials, n = 292). In cohort studies, one daily serving increment of SSBs was associated with a 0.06 (95% CI: 0.02, 0.10) and 0.05 (95% CI: 0.03, 0.07)-unit increase in BMI in children and 0.22 kg (95% CI: 0.09, 0.34 kg) and 0.12 kg (95% CI: 0.10, 0.14 kg) weight gain in adults over 1 y in random- and fixed-effects models, respectively. RCTs in children showed reductions in BMI gain when SSBs were reduced [random and fixed effects: -0.17 (95% CI: -0.39, 0.05) and -0.12 (95% CI: -0.22, -0.2)], whereas RCTs in adults showed increases in body weight when SSBs were added (random and fixed effects: 0.85 kg; 95% CI: 0.50, 1.20 kg). Sensitivity analyses of RCTs in children showed more pronounced benefits in preventing weight gain in SSB substitution trials (compared with school-based educational programs) and among overweight children (compared with normal-weight children). CONCLUSION Our systematic review and meta-analysis of prospective cohort studies and RCTs provides evidence that SSB consumption promotes weight gain in children and adults.


title: The tumorigenic potential of pluripotent stem cells: What can we do to minimize it?

text: Human pluripotent stem cells (hPSCs) have the potential to fundamentally change the way that we go about treating and understanding human disease. Despite this extraordinary potential, these cells also have an innate capability to form tumors in immunocompromised individuals when they are introduced in their pluripotent state. Although current therapeutic strategies involve transplantation of only differentiated hPSC derivatives, there is still a concern that transplanted cell populations could contain a small percentage of cells that are not fully differentiated. In addition, these cells have been frequently reported to acquire genetic alterations that, in some cases, are associated with certain types of human cancers. Here, we try to separate the panic from reality and rationally evaluate the true tumorigenic potential of these cells. We also discuss a recent study examining the effect of culture conditions on the genetic integrity of hPSCs. Finally, we present a set of sensible guidelines for minimizing the tumorigenic potential of hPSC-derived cells. © 2016 The Authors. Inside the Cell published by Wiley Periodicals, Inc.


title: Epidemiology of sepsis and septic shock in critical care units: comparison between sepsis-2 and sepsis-3 populations using a national critical care database

text: Background New sepsis and septic shock definitions could change the epidemiology of sepsis because of differences in criteria. We therefore compared the sepsis populations identified by the old and new definitions. Methods We used a high-quality, national, intensive care unit (ICU) database of 654 918 consecutive admissions to 189 adult ICUs in England, from January 2011 to December 2015. Primary outcome was acute hospital mortality. We compared old (Sepsis-2) and new (Sepsis-3) incidence, outcomes, trends in outcomes, and predictive validity of sepsis and septic shock populations. Results From among 197 724 Sepsis-2 severe sepsis and 197 142 Sepsis-3 sepsis cases, we identified 153 257 Sepsis-2 septic shock and 39 262 Sepsis-3 septic shock cases. The extrapolated population incidence of Sepsis-3 sepsis and Sepsis-3 septic shock was 101.8 and 19.3 per 100 000 person-years, respectively, in 2015. Sepsis-2 severe sepsis and Sepsis-3 sepsis had similar incidence, similar mortality and showed significant risk-adjusted improvements in mortality over time. Sepsis-3 septic shock had a much higher Acute Physiology And Chronic Health Evaluation II (APACHE II) score, greater mortality and no risk-adjusted trends in mortality improvement compared with Sepsis-2 septic shock. ICU admissions identified either as Sepsis-3 sepsis or septic shock and as Sepsis-2 severe sepsis or septic shock had significantly greater risk-adjusted odds of death compared with non-sepsis admissions (P<0.001). The predictive validity was greatest for Sepsis-3 septic shock. Conclusions In an ICU database, compared with Sepsis-2, Sepsis-3 identifies a similar sepsis population with 92% overlap and much smaller septic shock population with improved predictive validity.


title: Thymic expression of the golli-myelin basic protein gene in the SJL/J mouse

text: The T cell antigen-specific repertoire is thought to be shaped by thymic expression of self molecules. Since a myelin basic protein (MBP)-like gene (golli-MBP) has been reported to be expressed by cells of the immune system, the present study was undertaken to determine whether the golli-MBP gene was expressed in the mouse thymus and, if so, to characterize transcripts of this gene in this organ. Using exon-specific primers for MBP and golli-MBP, cDNA from thymus and other tissues was amplified, and the amplified products analyzed by Southern blotting with exon-specific oligonucleotide probes. The amplified products were subcloned, and the inserts characterized by DNA sequencing. The thymic transcripts were found to contain golli-MBP exons 1, 2, 3, 5A, 5B, 5C, 6, 7, 8, and 11.


title: Urine microRNAs as biomarkers for bladder cancer: a diagnostic meta-analysis

text: BACKGROUND The diagnostic value of microRNA (miRNA) detection in patients with bladder cancer (BCa) is controversial. We performed a diagnostic meta-analysis to evaluate current evidence on the use of miRNA assays to diagnose BCa. METHODS We systematically searched PubMed, Embase, and Web of Science for studies published before March 31, 2015. The pooled sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and area under the curve (AUC) were calculated to evaluate the overall test performance. Subgroup analyses were used to explore the between-study heterogeneity. Deeks' funnel plot asymmetry test was used to test publication bias. We applied the software of RevMan 5.2 and Stata 11.0 to the meta-analysis. RESULTS A total of 23 studies from nine articles were included in the meta-analysis, with a total of 719 patients and 494 controls. The pooled sensitivity and specificity were 0.75 (95% confidence interval [CI], 0.68-0.80) and 0.75 (95% CI, 0.70-0.80), respectively. The pooled positive likelihood ratio was 3.03 (95% CI, 2.50-3.67); negative likelihood ratio was 0.33 (95% CI, 0.27-0.42); and diagnostic odds ratio was 9.07 (95% CI, 6.35-12.95). The pooled AUC was 0.81 (95% CI, 0.78-0.85). Subgroup analyses indicated that the multiple miRNAs assays and urine supernatant assays showed high accuracies in diagnosing BCa. CONCLUSION The miRNA assays may serve as potential noninvasive diagnostic tool for the detection of BCa. However, the clinical application of miRNA assays for BCa diagnosis still needs further validation by large prospective studies.


title: Elevated Plasma Transforming Growth Factor &bgr;1 Levels Predict the Development of Hypertension in Normotensives: The 14-Year Follow-Up Study

text: BACKGROUND Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine. There is growing evidence that TGF-β1 is involved in the pathogenesis of hypertension and the development of target organ damage in hypertensives. Although several studies have shown that TGF-β1 induced vascular hypertrophy and remodelling in various vascular diseases, there are no longitudinal data on hypertension in the epidemiological studies. The present study tested the hypothesis whether elevated TGF-β1 levels can predict the development of hypertension. METHODS In 2002-2004, 528 subjects received health examinations in Uku town, southwestern Japan. We examined blood pressure (BP), body mass index, and blood test. Data on fasting plasma TGF-β1 were obtained from 528 individuals. Of these, 149 normotensives (BP <140/90 mm Hg without antihypertensive medications) at baseline were followed-up for 14 years. RESULTS The receiver-operating characteristic curve was used and the calculated cutoff value was 8.9 ng/ml. Of 149 normotensives at baseline, 59 subjects developed hypertension. Plasma TGF-β1 levels were significantly associated with the development of hypertension after adjustment for confounding factors. To further examine the association between them, we performed logistic regression analysis. We divided the baseline plasma TGF-β1 levels into 2 groups using a cutoff value. The significant high odds ratio [3.582 (95% confidence interval, 1.025-12.525)] for the development of hypertension was found in the highest group of TGF-β1 level vs. the lowest group after adjustment for confounders. CONCLUSIONS This is the first report demonstrating the causal relationship between them. Elevated plasma TGF-β1 levels predicted the development of hypertension in normotensives in a population of community-dwelling Japanese.


title: MYC Disrupts the Circadian Clock and Metabolism in Cancer Cells.

text: The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERBα to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERBα expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.


title: Integrated maps in quail (Coturnix japonica) confirm the high degree of synteny conservation with chicken (Gallus gallus) despite 35 million years of divergence

text: BackgroundBy comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps. ResultsThe integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied. ConclusionIntegrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.


title: Cadherin activity is required for activity-induced spine remodeling

text: Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.


title: Aliskiren combined with losartan in type 2 diabetes and nephropathy.

text: BACKGROUND Diabetic nephropathy is the leading cause of end-stage renal disease in developed countries. We evaluated the renoprotective effects of dual blockade of the renin-angiotensin-aldosterone system by adding treatment with aliskiren, an oral direct renin inhibitor, to treatment with the maximal recommended dose of losartan (100 mg daily) and optimal antihypertensive therapy in patients who had hypertension and type 2 diabetes with nephropathy. METHODS We enrolled 599 patients in this multinational, randomized, double-blind study. After a 3-month, open-label, run-in period during which patients received 100 mg of losartan daily, patients were randomly assigned to receive 6 months of treatment with aliskiren (150 mg daily for 3 months, followed by an increase in dosage to 300 mg daily for another 3 months) or placebo, in addition to losartan. The primary outcome was a reduction in the ratio of albumin to creatinine, as measured in an early-morning urine sample, at 6 months. RESULTS The baseline characteristics of the two groups were similar. Treatment with 300 mg of aliskiren daily, as compared with placebo, reduced the mean urinary albumin-to-creatinine ratio by 20% (95% confidence interval, 9 to 30; P<0.001), with a reduction of 50% or more in 24.7% of the patients who received aliskiren as compared with 12.5% of those who received placebo (P<0.001). A small difference in blood pressure was seen between the treatment groups by the end of the study period (systolic, 2 mm Hg lower [P=0.07] and diastolic, 1 mm Hg lower [P=0.08] in the aliskiren group). The total numbers of adverse and serious adverse events were similar in the groups. CONCLUSIONS Aliskiren may have renoprotective effects that are independent of its blood-pressure-lowering effect in patients with hypertension, type 2 diabetes, and nephropathy who are receiving the recommended renoprotective treatment. (ClinicalTrials.gov number, NCT00097955 [ClinicalTrials.gov].).


title: Overweight in Celiac Disease: Prevalence, Clinical Characteristics, and Effect of a Gluten-Free Diet

text: BACKGROUND:It is well established that a minority of celiac patients present with “classic” symptoms due to malabsorption. However, few studies have focussed on the distribution of body mass index (BMI) in celiac populations and its relationship to clinical characteristics, or on its response to treatment. METHODS:We reviewed BMI measurements and other clinical and pathological characteristics from a database of 371 celiac patients diagnosed over a 10-yr period and seen by a single gastroenterologist. To assess response to gluten exclusion, we compared BMI at diagnosis and after 2 yr treatment in patients with serological support for dietary compliance. RESULTS:Mean BMI was 24.6 kg/m2 (range 16.3–43.5). Seventeen patients (5%) were underweight (BMI <18.5), 211 (57%) were normal, and 143 (39%) were overweight (BMI ≥25), including 48 (13% of all patients) in the obese range (BMI ≥30.0). There was a significant association between low BMI and female gender, history of diarrhea, reduced hemoglobin concentration, reduced bone mineral density (BMD), osteoporosis, and higher grades (subtotal/total) of villous atrophy. Of patients compliant with a gluten-free diet, 81% had gained weight after 2 yr, including 82% of initially overweight patients. CONCLUSIONS:Few celiac patients are underweight at diagnosis and a large minority is overweight; these are less likely to present with classical features of diarrhea and reduced hemoglobin. Failed or delayed diagnosis of celiac disease may reflect lack of awareness of this large subgroup. The increase in weight of already overweight patients after dietary gluten exclusion is a potential cause of morbidity, and the gluten-free diet as conventionally prescribed needs to be modified accordingly.


title: Structural Studies of a Four-MBT Repeat Protein MBTD1

text: BACKGROUND The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats. METHODOLOGY/PRINCIPAL FINDINGS We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation. CONCLUSIONS The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage. ENHANCED VERSION This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.


title: Glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway

text: Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP(+) ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.


title: CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection.

text: Tissue-resident memory T (Trm) cells provide enhanced protection against infection at mucosal sites. Here we found that CD4(+) T cells are important for the formation of functional lung-resident CD8(+) T cells after influenza virus infection. In the absence of CD4(+) T cells, CD8(+) T cells displayed reduced expression of CD103 (Itgae), were mislocalized away from airway epithelia, and demonstrated an impaired ability to recruit CD8(+) T cells to the lung airways upon heterosubtypic challenge. CD4(+) T cell-derived interferon-γ was necessary for generating lung-resident CD103(+) CD8(+) Trm cells. Furthermore, expression of the transcription factor T-bet was increased in "unhelped" lung Trm cells, and a reduction in T-bet rescued CD103 expression in the absence of CD4(+) T cell help. Thus, CD4(+) T cell-dependent signals are important to limit expression of T-bet and allow for the development of CD103(+) CD8(+) Trm cells in the lung airways following respiratory infection.


title: Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

text: Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.


title: Computational and Statistical Analyses of Amino Acid Usage and Physico-Chemical Properties of the Twelve Late Embryogenesis Abundant Protein Classes

text: Late Embryogenesis Abundant Proteins (LEAPs) are ubiquitous proteins expected to play major roles in desiccation tolerance. Little is known about their structure - function relationships because of the scarcity of 3-D structures for LEAPs. The previous building of LEAPdb, a database dedicated to LEAPs from plants and other organisms, led to the classification of 710 LEAPs into 12 non-overlapping classes with distinct properties. Using this resource, numerous physico-chemical properties of LEAPs and amino acid usage by LEAPs have been computed and statistically analyzed, revealing distinctive features for each class. This unprecedented analysis allowed a rigorous characterization of the 12 LEAP classes, which differed also in multiple structural and physico-chemical features. Although most LEAPs can be predicted as intrinsically disordered proteins, the analysis indicates that LEAP class 7 (PF03168) and probably LEAP class 11 (PF04927) are natively folded proteins. This study thus provides a detailed description of the structural properties of this protein family opening the path toward further LEAP structure - function analysis. Finally, since each LEAP class can be clearly characterized by a unique set of physico-chemical properties, this will allow development of software to predict proteins as LEAPs.


title: Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

text: Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.


title: Epithelial to Mesenchymal Transition by TGFβ-1 Induction Increases Stemness Characteristics in Primary Non Small Cell Lung Cancer Cell Line

text: BACKGROUND Cancer Stem Cells (CSCs) hypothesis asserts that only a small subset of cells within a tumour is capable of both tumour initiation and sustainment. The Epithelial-Mesenchymal Transition (EMT) is an embryonic developmental program that is often activated during cancer invasion and metastasis. The aim of this study is to shed light on the relationship between EMT and CSCs by using LC31 lung cancer primary cell line. MATERIALS AND METHODS A549 and LC31 cell lines were treated with 2 ng/ml TGFβ-1 for 30 days, and 80 days, respectively. To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and cytometry for following markers: cytokeratins, e-cadherin, CD326 (epithelial markers) and CD90, and vimentin (mesenchymal markers). Moreover, RT-PCR for Slug, Twist and β-catenin genes were performed. On TGFβ-1 treated and untreated LC31 cell lines, we performed stemness tests such as pneumospheres growth and stem markers expression such as Oct4, Nanog, Sox2, c-kit and CD133. Western Blot for CD133 and tumorigenicity assays using NOD/SCID mice were performed. RESULTS TGFβ-1 treated LC31 cell line lost its epithelial morphology assuming a fibroblast-like appearance. The same results were obtained for the A549 cell line (as control). Immunofluorescence and cytometry showed up-regulation of vimentin and CD90 and down-regulation of cytocheratin, e-cadherin and CD326 in TGFβ-1 treated LC31 and A549 cell lines. Slug, Twist and β-catenin m-RNA transcripts were up-regulated in TGFβ-1 treated LC31 cell line confirming EMT. This cell line showed also over-expression of Oct4, Nanog, Sox2 and CD133, all genes of stemness. In addition, in TGFβ-1 treated LC31 cell line, an increased pneumosphere-forming capacity and tumours-forming ability in NOD/SCID mice were detectable. CONCLUSIONS The induction of EMT by TGFβ-1 exposure, in primary lung cancer cell line results in the acquisition of mesenchymal profile and in the expression of stem cell markers.


title: Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis.

text: The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.


title: CDK inhibitors in cancer therapy: what is next?

text: The pursuit for drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. The first-generation inhibitors, Flavopiridol and CY-202, are in late-stage clinical trials, but so far have demonstrated only modest activity. Several second-generation inhibitors are now in clinical trials. Future approaches to determine clinical benefit need to incorporate both the lessons learned from these early compounds and information recently obtained from the genetic analysis of CDKs in preclinical models. Here we discuss key concepts that should be considered when validating the clinical utility of CDK inhibitors in cancer therapy.


title: Lipid Desaturation Is a Metabolic Marker and Therapeutic Target of Ovarian Cancer Stem Cells.

text: Lack of sensitive single-cell analysis tools has limited the characterization of metabolic activity in cancer stem cells. By hyperspectral-stimulated Raman scattering imaging of single living cells and mass spectrometry analysis of extracted lipids, we report here significantly increased levels of unsaturated lipids in ovarian cancer stem cells (CSCs) as compared to non-CSCs. Higher lipid unsaturation levels were also detected in CSC-enriched spheroids compared to monolayer cultures of ovarian cancer cell lines or primary cells. Inhibition of lipid desaturases effectively eliminated CSCs, suppressed sphere formation in vitro, and blocked tumor initiation capacity in vivo. Mechanistically, we demonstrate that nuclear factor κB (NF-κB) directly regulates the expression levels of lipid desaturases, and inhibition of desaturases blocks NF-κB signaling. Collectively, our findings reveal that increased lipid unsaturation is a metabolic marker for ovarian CSCs and a target for CSC-specific therapy.


title: Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

text: Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.


title: Novel Functional Sets of Lipid-Derived Mediators with Antiinflammatory Actions Generated from Omega-3 Fatty Acids via Cyclooxygenase 2–Nonsteroidal Antiinflammatory Drugs and Transcellular Processing

text: Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with ω-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 ω-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX5 and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as ω-5 and ω-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2–nonsteroidal antiinflammatory drug–dependent oxygenations and cell–cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of ω-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.


title: Cholesteryl Ester Transfer Protein (CETP) Polymorphisms Affect mRNA Splicing, HDL Levels, and Sex-Dependent Cardiovascular Risk

text: Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.


title: 6-Sulphated Chondroitins Have a Positive Influence on Axonal Regeneration

text: Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.


title: Tissue Mechanics Orchestrate Wnt-Dependent Human Embryonic Stem Cell Differentiation.

text: Regenerative medicine is predicated on understanding the mechanisms regulating development and applying these conditions to direct stem cell fate. Embryogenesis is guided by cell-cell and cell-matrix interactions, but it is unclear how these physical cues influence stem cells in culture. We used human embryonic stem cells (hESCs) to examine whether mechanical features of the extracellular microenvironment could differentially modulate mesoderm specification. We found that, on a hydrogel-based compliant matrix, hESCs accumulate β-catenin at cell-cell adhesions and show enhanced Wnt-dependent mesoderm differentiation. Mechanistically, Src-driven ubiquitination of E-cadherin by Cbl-like ubiquitin ligase releases P120-catenin to facilitate transcriptional activity of β-catenin, which initiates and reinforces mesoderm differentiation. By contrast, on a stiff hydrogel matrix, hESCs show elevated integrin-dependent GSK3 and Src activity that promotes β-catenin degradation and inhibits differentiation. Thus, we found that mechanical features of the microenvironmental matrix influence tissue-specific differentiation of hESCs by altering the cellular response to morphogens.


title: Predictive performance of the quick Sequential Organ Failure Assessment score as a screening tool for sepsis, mortality, and intensive care unit admission in patients with febrile neutropenia

text: In Sepsis-3, the quick Sequential Organ Failure Assessment (qSOFA) score was developed as criteria to use for recognizing patients who may have poor outcomes. This study was performed to evaluate the predictive performance of the qSOFA score as a screening tool for sepsis, mortality, and intensive care unit (ICU) admission in patients with febrile neutropenia (FN). We also tried to compare its performance with that of the systemic inflammatory response syndrome (SIRS) criteria and Multinational Association of Supportive Care in Cancer (MASCC) score for FN. We used a prospectively collected adult FN data registry. The qSOFA and SIRS scores were calculated retrospectively using the preexisting data. The primary outcome was the development of sepsis. The secondary outcomes were ICU admission and 28-day mortality. Of the 615 patients, 100 developed sepsis, 20 died, and 38 were admitted to ICUs. In multivariate analysis, qSOFA was an independent factor predicting sepsis and ICU admission. However, compared to the MASCC score, the area under the receiver operating curve of qSOFA was lower. qSOFA showed a low sensitivity (0.14, 0.2, and 0.23) but high specificity (0.98, 0.97, and 0.97) in predicting sepsis, 28-day mortality, and ICU admission. Performance of the qSOFA score was inferior to that of the MASCC score. The preexisting risk stratification tool is more useful for predicting outcomes in patients with FN.


title: LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency.

text: The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.


title: Genetic Drift Can Compromise Mitochondrial Replacement by Nuclear Transfer in Human Oocytes.

text: Replacement of mitochondria through nuclear transfer between oocytes of two different women has emerged recently as a strategy for preventing inheritance of mtDNA diseases. Although experiments in human oocytes have shown effective replacement, the consequences of small amounts of mtDNA carryover have not been studied sufficiently. Using human mitochondrial replacement stem cell lines, we show that, even though the low levels of heteroplasmy introduced into human oocytes by mitochondrial carryover during nuclear transfer often vanish, they can sometimes instead result in mtDNA genotypic drift and reversion to the original genotype. Comparison of cells with identical oocyte-derived nuclear DNA but different mtDNA shows that either mtDNA genotype is compatible with the nucleus and that drift is independent of mitochondrial function. Thus, although functional replacement of the mitochondrial genome is possible, even low levels of heteroplasmy can affect the stability of the mtDNA genotype and compromise the efficacy of mitochondrial replacement.


title: TAL effectors are remote controls for gene activation.

text: TAL (transcription activator-like) effectors constitute a novel class of DNA-binding proteins with predictable specificity. They are employed by Gram-negative plant-pathogenic bacteria of the genus Xanthomonas which translocate a cocktail of different effector proteins via a type III secretion system (T3SS) into plant cells where they serve as virulence determinants. Inside the plant cell, TALs localize to the nucleus, bind to target promoters, and induce expression of plant genes. DNA-binding specificity of TALs is determined by a central domain of tandem repeats. Each repeat confers recognition of one base pair (bp) in the DNA. Rearrangement of repeat modules allows design of proteins with desired DNA-binding specificities. Here, we summarize how TAL specificity is encoded, first structural data and first data on site-specific TAL nucleases.


title: Vascular heterogeneity and specialization in development and disease

text: Blood and lymphatic vessels pervade almost all body tissues and have numerous essential roles in physiology and disease. The inner lining of these networks is formed by a single layer of endothelial cells, which is specialized according to the needs of the tissue that it supplies. Whereas the general mechanisms of blood and lymphatic vessel development are being defined with increasing molecular precision, studies of the processes of endothelial specialization remain mostly descriptive. Recent insights from genetic animal models illuminate how endothelial cells interact with each other and with their tissue environment, providing paradigms for vessel type- and organ-specific endothelial differentiation. Delineating these governing principles will be crucial for understanding how tissues develop and maintain, and how their function becomes abnormal in disease.


title: Association of Intracerebral Hemorrhage Among Patients Taking Non–Vitamin K Antagonist vs Vitamin K Antagonist Oral Anticoagulants With In-Hospital Mortality

text: Importance Although non–vitamin K antagonist oral anticoagulants (NOACs) are increasingly used to prevent thromboembolic disease, there are limited data on NOAC-related intracerebral hemorrhage (ICH). Objective To assess the association between preceding oral anticoagulant use (warfarin, NOACs, and no oral anticoagulants [OACs]) and in-hospital mortality among patients with ICH. Design, Setting, and Participants Retrospective cohort study of 141 311 patients with ICH admitted from October 2013 to December 2016 to 1662 Get With The Guidelines–Stroke hospitals. Exposures Anticoagulation therapy before ICH, defined as any use of OACs within 7 days prior to hospital arrival. Main Outcomes and Measures In-hospital mortality. Results Among 141 311 patients with ICH (mean [SD] age, 68.3 [15.3] years; 48.1% women), 15 036 (10.6%) were taking warfarin and 4918 (3.5%) were taking NOACs preceding ICH, and 39 585 (28.0%) and 5783 (4.1%) were taking concomitant single and dual antiplatelet agents, respectively. Patients with prior use of warfarin or NOACs were older and had higher prevalence of atrial fibrillation and prior stroke. Acute ICH stroke severity (measured by the National Institutes of Health Stroke Scale) was not significantly different across the 3 groups (median, 9 [interquartile range, 2-21] for warfarin, 8 [2-20] for NOACs, and 8 [2-19] for no OACs). The unadjusted in-hospital mortality rates were 32.6% for warfarin, 26.5% for NOACs, and 22.5% for no OACs. Compared with patients without prior use of OACs, the risk of in-hospital mortality was higher among patients with prior use of warfarin (adjusted risk difference [ARD], 9.0% [97.5% CI, 7.9% to 10.1%]; adjusted odds ratio [AOR], 1.62 [97.5% CI, 1.53 to 1.71]) and higher among patients with prior use of NOACs (ARD, 3.3% [97.5% CI, 1.7% to 4.8%]; AOR, 1.21 [97.5% CI, 1.11-1.32]). Compared with patients with prior use of warfarin, patients with prior use of NOACs had a lower risk of in-hospital mortality (ARD, −5.7% [97.5% CI, −7.3% to −4.2%]; AOR, 0.75 [97.5% CI, 0.69 to 0.81]). The difference in mortality between NOAC-treated patients and warfarin-treated patients was numerically greater among patients with prior use of dual antiplatelet agents (32.7% vs 47.1%; ARD, −15.0% [95.5% CI, −26.3% to −3.8%]; AOR, 0.50 [97.5% CI, 0.29 to 0.86]) than among those taking these agents without prior antiplatelet therapy (26.4% vs 31.7%; ARD, −5.0% [97.5% CI, −6.8% to −3.2%]; AOR, 0.77 [97.5% CI, 0.70 to 0.85]), although the interaction P value (.07) was not statistically significant. Conclusions and Relevance Among patients with ICH, prior use of NOACs or warfarin was associated with higher in-hospital mortality compared with no OACs. Prior use of NOACs, compared with prior use of warfarin, was associated with lower risk of in-hospital mortality.


title: Elevated red blood cell distribution width contributes to poor prognosis in patients undergoing resection for nonmetastatic rectal cancer

text: Several studies have reported that elevated red blood cell distribution width (RDW) was associated with the poor prognosis of different kinds of cancers. The aim of this study was to investigate the prognostic role of RDW in patients undergoing resection for nonmetastatic rectal cancer. We retrospectively reviewed a database of 625 consecutive patients who underwent curative resection for nonmetastatic rectal cancer at our institution from January 2009 to December 2014. The cutoff value of RDW was calculated by receiver-operating characteristic curve. The results demonstrated that patients in high RDW-cv group had a lower overall survival (OS) (P = .018) and disease-free survival (P = .004). We also observed that patients in high RDW-sd group were associated with significantly lower OS (P = .033), whereas the disease-free survival (DFS) was not significantly different (P = .179).In multivariate analysis, we found elevated RDW-cv was associated poor DFS (hazard ratio [HR] = 1.56, P = .010) and RDW-sd can predict a worse OS (HR = 1.70, P = .009).We confirmed that elevated RDW can be an independently prognostic factor in patients undergoing resection for nonmetastatic rectal cancer. So more intervention or surveillance might be paid to the patients with nonmetastatic rectal cancer and elevated RDW values in the future.


title: Health workers cohort study: methods and study design.

text: Objective: To examine different health outcomes that are associated with specific lifestyle and genetic factors. Materials and methods: From March 2004 to April 2006, a sample of employees from three different health and academic institutions, as well as their family members, were enrolled in the study after providing informed consent. At baseline and follow-up (2010-2013), participants completed a self-administered questionnaire, a physical examination, and provided blood samples. Results: A total of 10 729 participants aged 6 to 94 years were recruited at baseline. Of these, 70% were females, and 50% were from the Mexican Social Security Institute. Nearly 42% of the adults in the sample were overweight, while 20% were obese. Conclusion: Our study can offer new insights into disease mechanisms and prevention through the analysis of risk factor information in a large sample of Mexicans.


title: Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

text: OBJECTIVE Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. STUDY DESIGN Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. RESULTS Corin concentrations were significantly higher in mild (2.78 ± 0.67 ng/ml, p < .05) and severe (2.53 ± 0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ± 0.08 ng/ml) and CHT (1.55 ± 0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ± 0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ± 0.24 ng/ml, p < .01) than in normotensive (0.48 ± 0.06 ng/ml) and mild preeclampsia (0.52 ± 0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. CONCLUSIONS ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.


title: Optimising Strategies for Plasmodium falciparum Malaria Elimination in Cambodia: Primaquine, Mass Drug Administration and Artemisinin Resistance

text: BACKGROUND Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria. METHOD AND FINDINGS A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity. CONCLUSIONS The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination.


title: Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection

text: Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.


title: Axicabtagene Ciloleucel CAR T‐Cell Therapy in Refractory Large B‐Cell Lymphoma

text: Background In a phase 1 trial, axicabtagene ciloleucel (axi‐cel), an autologous anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy, showed efficacy in patients with refractory large B‐cell lymphoma after the failure of conventional therapy. Methods In this multicenter, phase 2 trial, we enrolled 111 patients with diffuse large B‐cell lymphoma, primary mediastinal B‐cell lymphoma, or transformed follicular lymphoma who had refractory disease despite undergoing recommended prior therapy. Patients received a target dose of 2×106 anti‐CD19 CAR T cells per kilogram of body weight after receiving a conditioning regimen of low‐dose cyclophosphamide and fludarabine. The primary end point was the rate of objective response (calculated as the combined rates of complete response and partial response). Secondary end points included overall survival, safety, and biomarker assessments. Results Among the 111 patients who were enrolled, axi‐cel was successfully manufactured for 110 (99%) and administered to 101 (91%). The objective response rate was 82%, and the complete response rate was 54%.With a median follow‐up of 15.4 months, 42% of the patients continued to have a response, with 40% continuing to have a complete response. The overall rate of survival at 18 months was 52%. The most common adverse events of grade 3 or higher during treatment were neutropenia (in 78% of the patients), anemia (in 43%), and thrombocytopenia (in 38%). Grade 3 or higher cytokine release syndrome and neurologic events occurred in 13% and 28% of the patients, respectively. Three of the patients died during treatment. Higher CAR T‐cell levels in blood were associated with response. Conclusions In this multicenter study, patients with refractory large B‐cell lymphoma who received CAR T‐cell therapy with axi‐cel had high levels of durable response, with a safety profile that included myelosuppression, the cytokine release syndrome, and neurologic events. (Funded by Kite Pharma and the Leukemia and Lymphoma Society Therapy Acceleration Program; ZUMA‐1 ClinicalTrials.gov number, NCT02348216.)


title: Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

text: During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.


title: Stereotactic radiosurgery plus whole-brain radiation therapy vs stereotactic radiosurgery alone for treatment of brain metastases: a randomized controlled trial.

text: CONTEXT In patients with brain metastases, it is unclear whether adding up-front whole-brain radiation therapy (WBRT) to stereotactic radiosurgery (SRS) has beneficial effects on mortality or neurologic function compared with SRS alone. OBJECTIVE To determine if WBRT combined with SRS results in improvements in survival, brain tumor control, functional preservation rate, and frequency of neurologic death. DESIGN, SETTING, AND PATIENTS Randomized controlled trial of 132 patients with 1 to 4 brain metastases, each less than 3 cm in diameter, enrolled at 11 hospitals in Japan between October 1999 and December 2003. INTERVENTIONS Patients were randomly assigned to receive WBRT plus SRS (65 patients) or SRS alone (67 patients). MAIN OUTCOME MEASURES The primary end point was overall survival; secondary end points were brain tumor recurrence, salvage brain treatment, functional preservation, toxic effects of radiation, and cause of death. RESULTS The median survival time and the 1-year actuarial survival rate were 7.5 months and 38.5% (95% confidence interval, 26.7%-50.3%) in the WBRT + SRS group and 8.0 months and 28.4% (95% confidence interval, 17.6%-39.2%) for SRS alone (P = .42). The 12-month brain tumor recurrence rate was 46.8% in the WBRT + SRS group and 76.4% for SRS alone group (P<.001). Salvage brain treatment was less frequently required in the WBRT + SRS group (n = 10) than with SRS alone (n = 29) (P<.001). Death was attributed to neurologic causes in 22.8% of patients in the WBRT + SRS group and in 19.3% of those treated with SRS alone (P = .64). There were no significant differences in systemic and neurologic functional preservation and toxic effects of radiation. CONCLUSIONS Compared with SRS alone, the use of WBRT plus SRS did not improve survival for patients with 1 to 4 brain metastases, but intracranial relapse occurred considerably more frequently in those who did not receive WBRT. Consequently, salvage treatment is frequently required when up-front WBRT is not used. TRIAL REGISTRATION umin.ac.jp/ctr Identifier: C000000412.


title: RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

text: Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.


title: Proteomic approach to characterize biochemistry of meat quality defects.

text: Proteomics can be used to characterize quality defects including pale, soft, and exudative (PSE) meat (pork and poultry), woody broiler breast meat, reddish catfish fillets, meat toughness, and beef myoglobin oxidation. PSE broiler meat was characterized by 15 proteins that differed in abundance in comparison to normal broiler breast meat, and eight proteins were differentially expressed in woody breast meat in comparison to normal breast meat. Hemoglobin was the only protein that was differentially expressed between red and normal catfish fillets. However, inducing low oxygen and/or heat stress conditions to catfish fillets did not lead to the production of red fillets. Proteomic data provided information pertaining to the protein differences that exist in meat quality defects. However, these data need to be evaluated in conjunction with information pertaining to genetics, nutrition, environment of the live animal, muscle to meat conversion, meat quality analyses and sensory attributes to understand causality, protein biomarkers, and ultimately how to prevent quality defects.


title: Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling

text: Adenosine 5′-monophosphate–activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.


title: IAP antagonists induce anti-tumor immunity in multiple myeloma

text: The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-κB pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.


title: Psychobiological Protective Factors Modifying the Association Between Age and Sexual Health in Men: Findings From the Men’s Health 40+ Study

text: Sexual health severely decreases with age. For males older than 40 years, erectile dysfunction (ED) is the most common sexual disorder. Although physical and psychological risk factors for ED have been identified, protective factors are yet to be determined. To date, no study has examined endocrine and psychosocial factors in parallel with regard to their modifying effect on the age-related increase in ED. Two hundred and seventy-one self-reporting healthy men aged between 40 and 75 years provided both psychometric data on sexual function and a set of potential psychosocial protective factors, and saliva samples for the analysis of steroid hormones and proinflammatory cytokines. Around 35% of the participants reported at least a mild form of ED. Direct associations with ED were identified for perceived general health, emotional support, relationship quality, intimacy motivation but not for steroid hormones or proinflammatory markers. Moderation analyses for the association between age and ED revealed positive effects for testosterone (T), dehydroepiandrosterone (DHEA), perceived general health, emotional support, intimacy motivation, and a negative effect for interleukin-6 (all p < .05; f2 > .17). Group differences between older men with and without ED emerged for T, DHEA, and psychometric measures such as perceived general health, emotional support, satisfaction with life, and intimacy motivation (all p < .05; d > .3). Both psychosocial and endocrine parameters moderated the association between age and sexual health. Perceived general health, emotional support, intimacy motivation, and relationship quality emerged as psychosocial protective factors against ED. Higher T and DHEA and lower interleukin-6 levels also buffered against an age-related increase in ED.


title: Patient-derived induced pluripotent stem cells in cancer research and precision oncology

text: Together with recent advances in the processing and culture of human tissue, bioengineering, xenotransplantation and genome editing, Induced pluripotent stem cells (iPSCs) present a range of new opportunities for the study of human cancer. Here we discuss the main advantages and limitations of iPSC modeling, and how the method intersects with other patient-derived models of cancer, such as organoids, organs-on-chips and patient-derived xenografts (PDXs). We highlight the opportunities that iPSC models can provide beyond those offered by existing systems and animal models and present current challenges and crucial areas for future improvements toward wider adoption of this technology.


title: Structural basis for sequence-specific recognition of DNA by TAL effectors.

text: TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.


title: Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

text: Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-β signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.


title: Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method

text: Study of the role of hydroperoxides and lipid peroxidation in disease requires simple and sensitive methods for direct hydroperoxide measurement. We report on a technique for measuring hydroperoxide which relies upon the rapid hydroperoxide-mediated oxidation of Fe2+ under acidic conditions. Fe3+ forms a chromophore with xylenol orange which absorbs strongly at 560 nm, yielding an apparent E560 (for H2O2, butyl hydroperoxide and cumene hydroperoxide) of 4.3×104 M−1 cm−1. The assay was validated in a study of liposomal lipid peroxidation and shown to give results comparable with those obtained by an iodometric method or by measuring conjugated dienes. The assay involving thiobarbituric acid, by comparison, underestimates lipid peroxidation and does not measure hydroperoxideper se.


title: Roles of exosomes in cardioprotection.

text: Exosomes are extracellular vesicles of endosomal origin which have emerged as key mediators of intercellular communication. All major cardiac cell types-including cardiomyocytes, endothelial cells, and fibroblasts-release exosomes that modulate cellular functions. Exosomes released from human cardiac progenitor cells (CPCs) are cardioprotective and improve cardiac function after myocardial infarction to an extent comparable with that achieved by their parent cells. Cardiac progenitor cell-derived exosomes are enriched in cardioprotective microRNAs, particularly miR-146a-3p. Circulating exosomes mediate remote ischaemic preconditioning. Moreover, they currently are being investigated as diagnostic markers. The discovery that cell-derived extracellular signalling organelles mediate the paracrine effects of stem cells suggests that cell-free strategies could supplant cell transplantation. This review discusses emerging roles of exosomes in cardiovascular physiology, with a focus on cardioprotective activities of CPC-derived exosomes.


title: The rate of change in declining steroid hormones: a new parameter of healthy aging in men?

text: Research on healthy aging in men has increasingly focused on age-related hormonal changes. Testosterone (T) decline is primarily investigated, while age-related changes in other sex steroids (dehydroepiandrosterone [DHEA], estradiol [E2], progesterone [P]) are mostly neglected. An integrated hormone parameter reflecting aging processes in men has yet to be identified. 271 self-reporting healthy men between 40 and 75 provided both psychometric data and saliva samples for hormone analysis. Correlation analysis between age and sex steroids revealed negative associations for the four sex steroids (T, DHEA, E2, and P). Principal component analysis including ten salivary analytes identified a principal component mainly unifying the variance of the four sex steroid hormones. Subsequent principal component analysis including the four sex steroids extracted the principal component of declining steroid hormones (DSH). Moderation analysis of the association between age and DSH revealed significant moderation effects for psychosocial factors such as depression, chronic stress and perceived general health. In conclusion, these results provide further evidence that sex steroids decline in aging men and that the integrated hormone parameter DSH and its rate of change can be used as biomarkers for healthy aging in men. Furthermore, the negative association of age and DSH is moderated by psychosocial factors.


title: Development of a novel helper-dependent adenovirus-Epstein-Barr virus hybrid system for the stable transformation of mammalian cells.

text: Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.


title: Spliceosome assembly pathways for different types of alternative splicing converge during commitment to splice site pairing in the A complex.

text: Differential splice site pairing establishes alternative splicing patterns resulting in the generation of multiple mRNA isoforms. This process is carried out by the spliceosome, which is activated by a series of sequential structural rearrangements of its five core snRNPs. To determine when splice sites become functionally paired, we carried out a series of kinetic trap experiments using pre-mRNAs that undergo alternative 5' splice site selection or alternative exon inclusion. We show that commitment to splice site pairing in both cases occurs in the A complex, which is characterized by the ATP-dependent association of the U2 snRNP with the branch point. Interestingly, the timing of splice site pairing is independent of the intron or exon definition modes of splice site recognition. Using the ATP analog ATPgammaS, we showed that ATP hydrolysis is required for splice site pairing independent from U2 snRNP binding to the pre-mRNA. These results identify the A complex as the spliceosomal assembly step dedicated to splice site pairing and suggest that ATP hydrolysis locks splice sites into a splicing pattern after stable U2 snRNP association to the branch point.


title: Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

text: The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.


title: Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells

text: Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells through large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown. Here we show that Ras-transformed cells use macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of nutrient uptake in tumours, its pharmacological inhibition compromises the growth of Ras-transformed pancreatic tumour xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer therapies.


title: Linking Splicing to Pol II Transcription Stabilizes Pre-mRNAs and Influences Splicing Patterns

text: RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.


title: The emerging role of skeletal muscle extracellular matrix remodelling in obesity and exercise.

text: Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise.