Dosing convenience is a key element in the effective management of any chronic disease, and is particularly important in the long-term management of osteoporosis. Less frequent dosing with any medication may enhance compliance, thereby maximizing the effectiveness of therapy. Animal data support the rationale that once-weekly dosing with alendronate 70 mg (7 times the daily oral treatment dose) could provide similar efficacy to daily dosing with alendronate 10 mg due to its long duration of effect in bone. In addition, dog studies suggest that the potential for esophageal irritation, observed with daily oral bisphosphonates, may be substantially reduced with once-weekly dosing. This dosing regimen would provide patients with increased convenience and would be likely to enhance patient compliance. We compared the efficacy and safety of treatment with oral once-weekly alendronate 70 mg (N=519), twice-weekly alendronate 35 mg (N=369), and daily alendronate 10 mg (N=370) in a one-year, double-blind, multicenter study of postmenopausal women (ages 42 to 95) with osteoporosis (bone mineral density [BMD] of either lumbar spine or femoral neck at least 2.5 SDs below peak premenopausal mean, or prior vertebral or hip fracture). The primary efficacy endpoint was the comparability of increases in lumbar spine BMD, using strict pre-defined equivalence criteria. Secondary endpoints included changes in BMD at the hip and total body and rate of bone turnover, as assessed by biochemical markers. Both of the new regimens fully satisfied the equivalence criteria relative to daily therapy. Mean increases in lumbar spine BMD at 12 months were: 5.1% (95% CI 4.8, 5.4) in the 70 mg once-weekly group, 5.2% (4.9, 5.6) in the 35 mg twice-weekly group, and 5.4% (5.0, 5.8) in the 10 mg daily treatment group. Increases in BMD at the total hip, femoral neck, trochanter, and total body were similar for the three dosing regimens. All three treatment groups similarly reduced biochemical markers of bone resorption (urinary N-telopeptides of type I collagen) and bone formation (serum bone-specific alkaline phosphatase) into the middle of the premenopausal reference range. All treatment regimens were well tolerated with a similar incidence of upper GI adverse experiences. There were fewer serious upper GI adverse experiences and a trend toward a lower incidence of esophageal events in the once-weekly dosing group compared to the daily dosing group. These data are consistent with preclinical animal models, and suggest that once-weekly dosing has the potential for improved upper GI tolerability. Clinical fractures, captured as adverse experiences, were similar among the groups. We conclude that the alendronate 70 mg once-weekly dosing regimen will provide patients with a more convenient, therapeutically equivalent alternative to daily dosing, and may enhance compliance and long-term persistence with therapy.


BACKGROUND Although Toll-like receptor 4 (TLR-4) is involved in monocyte activation in patients with accelerated forms of atherosclerosis, the relationship between the expression of TLR-4 on circulating monocytes and coronary plaque vulnerability has not previously been evaluated. We investigated this relationship using 64-slice multidetector computed tomography (MDCT) in patients with stable angina pectoris (SAP).Methods and Results:We enrolled 65 patients with SAP who underwent MDCT. Three monocyte subsets (CD14++CD16-, CD14++CD16+, and CD14+CD16+) and expression of TLR-4 were measured by flow cytometry. Intracoronary plaques were assessed by 64-slice MDCT. We defined vulnerability of intracoronary plaques according to the presence of positive remodeling (remodeling index >1.05) and/or low CT attenuation (<35 HU). The circulating CD14++CD16+monocytes more frequently expressed TLR-4 than CD14++CD16-and CD14+CD16+monocytes (P<0.001). The relative proportion of the expression of TLR-4 on CD14++CD16+monocytes was significantly greater in patients with vulnerable plaque compared with those without (10.4 [4.1-14.5] % vs. 4.5 [2.8-7.8] %, P=0.012). In addition, the relative proportion of TLR-4 expression on CD14++CD16+monocytes positively correlated with the remodeling index (r=0.28, P=0.025) and negatively correlated with CT attenuation value (r=-0.31, P=0.013). CONCLUSIONS Upregulation of TLR-4 on CD14++CD16+monocytes might be associated with coronary plaque vulnerability in patients with SAP.


Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.


Driven by technological progress, human life expectancy has increased greatly since the nineteenth century. Demographic evidence has revealed an ongoing reduction in old-age mortality and a rise of the maximum age at death, which may gradually extend human longevity. Together with observations that lifespan in various animal species is flexible and can be increased by genetic or pharmaceutical intervention, these results have led to suggestions that longevity may not be subject to strict, species-specific genetic constraints. Here, by analysing global demographic data, we show that improvements in survival with age tend to decline after age 100, and that the age at death of the world’s oldest person has not increased since the 1990s. Our results strongly suggest that the maximum lifespan of humans is fixed and subject to natural constraints.


OBJECTIVE To estimate the lost investment of domestically educated doctors migrating from sub-Saharan African countries to Australia, Canada, the United Kingdom, and the United States. DESIGN Human capital cost analysis using publicly accessible data. SETTINGS Sub-Saharan African countries. PARTICIPANTS Nine sub-Saharan African countries with an HIV prevalence of 5% or greater or with more than one million people with HIV/AIDS and with at least one medical school (Ethiopia, Kenya, Malawi, Nigeria, South Africa, Tanzania, Uganda, Zambia, and Zimbabwe), and data available on the number of doctors practising in destination countries. MAIN OUTCOME MEASURES The financial cost of educating a doctor (through primary, secondary, and medical school), assuming that migration occurred after graduation, using current country specific interest rates for savings converted to US dollars; cost according to the number of source country doctors currently working in the destination countries; and savings to destination countries of receiving trained doctors. RESULTS In the nine source countries the estimated government subsidised cost of a doctor's education ranged from $21,000 (£13,000; €15,000) in Uganda to $58,700 in South Africa. The overall estimated loss of returns from investment for all doctors currently working in the destination countries was $2.17bn (95% confidence interval 2.13bn to 2.21bn), with costs for each country ranging from $2.16m (1.55m to 2.78m) for Malawi to $1.41bn (1.38bn to 1.44bn) for South Africa. The ratio of the estimated compounded lost investment over gross domestic product showed that Zimbabwe and South Africa had the largest losses. The benefit to destination countries of recruiting trained doctors was largest for the United Kingdom ($2.7bn) and United States ($846m). CONCLUSIONS Among sub-Saharan African countries most affected by HIV/AIDS, lost investment from the emigration of doctors is considerable. Destination countries should consider investing in measurable training for source countries and strengthening of their health systems.


The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases. Both environmental factors and genetic factors influence human immunity. Albert and colleagues leverage data from the Milieu Intérieur Consortium to comprehensively describe the effects of lifestyle, environment and genetics on human innate and adaptive immunity.


Mast cells are unique tissue-resident immune cells that express an array of receptors that can be activated by several extracellular cues, including antigen-immunoglobulin E (IgE) complexes, bacteria, viruses, cytokines, hormones, peptides, and drugs. Mast cells constitute a small population in tissues, but their extraordinary ability to respond rapidly by releasing granule-stored and newly made mediators underpins their importance in health and disease. In this review, we document the biology of mast cells and introduce new concepts and opinions regarding their role in human diseases beyond IgE-mediated allergic responses and antiparasitic functions. We bring to light recent discoveries and developments in mast cell research, including regulation of mast cell functions, differentiation, survival, and novel mouse models. Finally, we highlight the current and future opportunities for therapeutic intervention of mast cell functions in inflammatory diseases.


LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.


The standard treatment of partial-thickness burns includes topical silver products such as silver sulfadiazine (SSD) cream and enclosed dressings including silver-impregnated foam (Mepilex Ag; Molnlycke Health Care, Gothenburg, Sweden) and silver-laden sheets (Aquacel Ag; ConvaTec, Skillman, NJ). The current state of health care is limited by resources, with an emphasis on evidence-based outcomes and cost-effective treatments. This study includes a decision analysis with an incremental cost-utility ratio comparing enclosed silver dressings with SSD in partial-thickness burn patients with TBSA less than 20%. A comprehensive literature review was conducted to identify clinically relevant health states in partial-thickness burn patients. These health states include successful healing, infection, and noninfected delayed healing requiring either surgery or conservative management. The probabilities of these health states were combined with Medicare CPT reimbursement codes (cost) and patient-derived utilities to fit into the decision model. Utilities were obtained using a visual analog scale during patient interviews. Expected cost and quality-adjusted life years (QALYs) were calculated using the roll-back method. The incremental cost-utility ratio for enclosed silver dressing relative to SSD was $40,167.99/QALY. One-way sensitivity analysis of complication rates confirmed robustness of the model. Assuming a maximum willingness to pay $50,000/QALY, the complication rate for SSD must be 22% or higher for enclosed silver dressing to be cost effective. By varying complication rates for SSD and enclosed silver dressings, the two-way sensitivity analysis demonstrated the cost effectiveness of using enclosed silver dressing at the majority of complication rates for both treatment modalities. Enclosed silver dressings are a cost-effective means of treating partial thickness burns.


This prospective, randomized study compared protocols of care using either AQUACEL Ag Hydrofiber (ConvaTec, a Bristol-Myers Squibb company, Skillman, NJ) dressing with silver (n = 42) or silver sulfadiazine (n = 42) for up to 21 days in the management of partial-thickness burns covering 5% to 40% body surface area (BSA). AQUACEL Ag dressing was associated with less pain and anxiety during dressing changes, less burning and stinging during wear, fewer dressing changes, less nursing time, and fewer procedural medications. Silver sulfadiazine was associated with greater flexibility and ease of movement. Adverse events, including infection, were comparable between treatment groups. The AQUACEL Ag dressing protocol tended to have lower total treatment costs (Dollars 1040 vs. Dollars 1180) and a greater rate of re-epithelialization (73.8% vs 60.0%), resulting in cost-effectiveness per burn healed of Dollars 1,409.06 for AQUACEL Ag dressing and Dollars 1,967.95 for silver sulfadiazine. A protocol of care with AQUACEL(R) Ag provided clinical and economic benefits compared with silver sulfadiazine in patients with partial-thickness burns.


The risk of HCV transmission by blood and blood products has been greatly reduced since the early 1980's. Selection of non-remunerated donors, donor selection to prevent HIV transmission, initial surrogate testing in some regions, and introduction of anti-HCV testing have all contributed to this. ALT surrogate testing has become obsolete since the introduction of anti-HCV testing. The residual risk of HCV transmission due to donations in the anti-HCV window period at present is about 1 in 100 000 transfusions of cellular products, and transmission of HCV by plasma products treated with modern inactivation methods such as solvent-detergent treatment, has not been reported. Hemovigilance programmes, which are presently being installed, will provide more data on the safety of blood transfusion. Introduction of HCV nucleic amplification technology (NAT) as a quality control of manufacturing pools for plasma products or as a form of blood donor screening by minipools is anticipated in many European countries for the coming year. Given industrial developments, NAT testing of individual blood donations may become available within the next 2 years. HCV NAT testing will further annihilate the residual risk, and the cost-effectiveness will become relatively low in comparison with other public health measures.


In the coming decades, a massive shift in the aging segment of the population will have major social and economic consequences around the world. One way to offset this increase is to expedite the development of geroprotectors, substances that slow aging, repair age-associated damage and extend healthy lifespan, or healthspan. While over 200 geroprotectors are now reported in model organisms and some are in human use for specific disease indications, the path toward determining whether they affect aging in humans remains obscure. Translation to the clinic is hampered by multiple issues including absence of a common set of criteria to define, select, and classify these substances, given the complexity of the aging process and their enormous diversity in mechanism of action. Translational research efforts would benefit from the formation of a scientific consensus on the following: the definition of 'geroprotector', the selection criteria for geroprotectors, a comprehensive classification system, and an analytical model. Here, we review current approaches to selection and put forth our own suggested selection criteria. Standardizing selection of geroprotectors will streamline discovery and analysis of new candidates, saving time and cost involved in translation to clinic.


β-catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway; alterations in its activity have been associated with the development of hepatocellular carcinoma and other liver diseases. Other than WNT, additional signaling pathways also can converge at β-catenin. β-catenin also interacts with transcription factors such as T-cell factor, forkhead box protein O, and hypoxia inducible factor 1α to regulate the expression of target genes. We discuss the role of β-catenin in metabolic zonation of the adult liver. β-catenin also regulates the expression of genes that control metabolism of glucose, nutrients, and xenobiotics; alterations in its activity may contribute to the pathogenesis of nonalcoholic steatohepatitis. Alterations in β-catenin signaling may lead to activation of hepatic stellate cells, which is required for fibrosis. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in CTNNB1 that result in constitutive activation of β-catenin, so this molecule could be a therapeutic target. We discuss how alterations in β-catenin activity contribute to liver disease and how these might be used in diagnosis and prognosis, as well as in the development of therapeutics.


BACKGROUND Dengue is the most common arbovirus infection globally, but its burden is poorly quantified. We estimated dengue mortality, incidence, and burden for the Global Burden of Disease Study 2013. METHODS We modelled mortality from vital registration, verbal autopsy, and surveillance data using the Cause of Death Ensemble Modelling tool. We modelled incidence from officially reported cases, and adjusted our raw estimates for under-reporting based on published estimates of expansion factors. In total, we had 1780 country-years of mortality data from 130 countries, 1636 country-years of dengue case reports from 76 countries, and expansion factor estimates for 14 countries. FINDINGS We estimated an average of 9221 dengue deaths per year between 1990 and 2013, increasing from a low of 8277 (95% uncertainty estimate 5353-10 649) in 1992, to a peak of 11 302 (6790-13 722) in 2010. This yielded a total of 576 900 (330 000-701 200) years of life lost to premature mortality attributable to dengue in 2013. The incidence of dengue increased greatly between 1990 and 2013, with the number of cases more than doubling every decade, from 8·3 million (3·3 million-17·2 million) apparent cases in 1990, to 58·4 million (23·6 million-121·9 million) apparent cases in 2013. When accounting for disability from moderate and severe acute dengue, and post-dengue chronic fatigue, 566 000 (186 000-1 415 000) years lived with disability were attributable to dengue in 2013. Considering fatal and non-fatal outcomes together, dengue was responsible for 1·14 million (0·73 million-1·98 million) disability-adjusted life-years in 2013. INTERPRETATION Although lower than other estimates, our results offer more evidence that the true symptomatic incidence of dengue probably falls within the commonly cited range of 50 million to 100 million cases per year. Our mortality estimates are lower than those presented elsewhere and should be considered in light of the totality of evidence suggesting that dengue mortality might, in fact, be substantially higher. FUNDING Bill & Melinda Gates Foundation.


Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.


Despite progress in treatment of small cell lung cancer (SCLC), its multidrug chemoresistance and poor prognosis still remain. Recently, we globally assessed long non-coding RNAs (lncRNAs) for contributions to SCLC chemoresistance using microarray data, in vitro and in vivo assays. Here we reported that HOTTIP, encoding a lncRNA that is frequently amplified in SCLC, was associated with SCLC cell chemosensitivity, proliferation, and poor prognosis of SCLC patients. Moreover, mechanistic investigations showed that HOTTIP functioned as an oncogene in SCLC progression by binding miR-216a and abrogating its tumor-suppressive function in this setting. On the other hand, HOTTIP increased the expression of anti-apoptotic factor BCL-2, another important target gene of miR-216a, and jointly enhanced chemoresistance of SCLC by regulating BCL-2. Taken together, our study established a role for HOTTIP in SCLC progression and chemoresistance suggest its candidacy as a new diagnostic and prognostic biomarker for clinical management of SCLC.


Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65–dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)–protein kinase B (PKB)–Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.)


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


INTERACTIONS between major histocompatibility complex (MHC) molecules and the CD4 or CDS coreceptors have a major role in intrathymic T-cell selection1. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay2–5, and a binding site for CDS on class I has been identified6,7. Here we demonstrate that a region of the MHC class IIβ-chain β2 domain, structurally analogous to the CDS-binding loop in the MHC class I α3 domain, is critical for function with both mouse and human CD4.


Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.


Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in the Western world. Twenty per cent of NAFLD individuals develop chronic hepatic inflammation (non-alcoholic steatohepatitis, NASH) associated with cirrhosis, portal hypertension and hepatocellular carcinoma, yet the causes of progression from NAFLD to NASH remain obscure. Here, we show that the NLRP6 and NLRP3 inflammasomes and the effector protein IL-18 negatively regulate NAFLD/NASH progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different mouse models reveal that inflammasome-deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, leading to enhanced hepatic tumour-necrosis factor (TNF)-α expression that drives NASH progression. Furthermore, co-housing of inflammasome-deficient mice with wild-type mice results in exacerbation of hepatic steatosis and obesity. Thus, altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of heretofore seemingly unrelated systemic auto-inflammatory and metabolic disorders.


Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process, and factors involved remain largely unknown. Here, we have identified the DEAD-box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability or 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis, and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that takes place mainly near the mitochondrial nucleoids, in the compartment defined by the RNA granules.


Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.


Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.


Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.


All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments. In the prevailing model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue. Acting via the β(3)-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes, whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.


Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.


The expression of clock genes in vertebrates is widespread and not restricted to classical clock structures. The expression of the Clock gene in zebrafish shows a strong circadian oscillation in many tissues in vivo and in culture, showing that endogenous oscillators exist in peripheral organs. A defining feature of circadian clocks is that they can be set or entrained to local time, usually by the environmental light-dark cycle. An important question is whether peripheral oscillators are entrained to local time by signals from central pacemakers such as the eyes or are themselves directly light-responsive. Here we show that the peripheral organ clocks of zebrafish are set by light-dark cycles in culture. We also show that a zebrafish-derived cell line contains a circadian oscillator, which is also directly light entrained.


The KRAS oncogene product is considered a major target in anticancer drug discovery. However, direct interference with KRAS signalling has not yet led to clinically useful drugs. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS–PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.


BCL-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma1–3. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths4–13. An emerging family of Bcl-2 -related proteins share two highly conserved regions14–20 referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-214. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitu-tion of Gly 145 in BHl domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in inter-leukin-3 deprivation, γ-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homo-dimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.


The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon. The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein. Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown. Here we show that TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2. The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3. Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4. TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2. Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm.


Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo1,2 which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage3, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals4 and a useful system for the identification of polypeptide factors controlling differentiation processes in early development5. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells6. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA7. Here, we report that purified DIA is related in structure and function to the recently identified haemopoetic regulatory factors human interleukin for DA cells8,9 and leukaemia inhibitory factor10. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and haemopoetic stem cell systems.


Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.


The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-α and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-α complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.


Despite decades of research, the identity of the cells generating the first haematopoietic cells in mammalian embryos is unknown. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial–haematopoietic precursors or haemogenic endothelial cells remains controversial. Proximity of endothelial and blood cells at sites of embryonic haematopoiesis, as well as their similar gene expression, led to the hypothesis of the endothelium generating blood. However, owing to lacking technology it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed. Here, using new imaging and cell-tracking methods, we show that embryonic endothelial cells can be haemogenic. By continuous long-term single-cell observation of mouse mesodermal cells generating endothelial cell and blood colonies, it was possible to detect haemogenic endothelial cells giving rise to blood cells. Living endothelial and haematopoietic cells were identified by simultaneous detection of morphology and multiple molecular and functional markers. Detachment of nascent blood cells from endothelium is not directly linked to asymmetric cell division, and haemogenic endothelial cells are specified from cells already expressing endothelial markers. These results improve our understanding of the developmental origin of mammalian blood and the potential generation of haematopoietic stem cells from embryonic stem cells.