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Abstract Background Sepsis from infection is a global health priority and clinical trials have failed to deliver effective therapeutic interventions. To address complicating heterogeneity in sepsis pathobiology, and improve outcomes, promising precision medicine approaches are helping identify disease endotypes, however, they require a more complete definition of sepsis subgroups. Methods Here, we use RNA sequencing from peripheral blood to interrogate the host response to sepsis from participants in a global observational study carried out in West Africa, Southeast Asia, and North America (N = 494). Results We identify four sepsis subtypes differentiated by 28-day mortality. A low mortality immunocompetent group is specified by features that describe the adaptive immune system. In contrast, the three high mortality groups show elevated clinical severity consistent with multiple organ dysfunction. The immunosuppressed group members show signs of a dysfunctional immune response, the acute-inflammation group is set apart by molecular features of the innate immune response, while the immunometabolic group is characterized by metabolic pathways such as heme biosynthesis. Conclusions Our analysis reveals details of molecular endotypes in sepsis that support immunotherapeutic interventions and identifies biomarkers that predict outcomes in these groups.

Abstract Oxycodone is one of the most widely prescribed and misused opioid painkillers in the United States. Evidence suggests that biological sex and hormonal status can impact drug reward in humans and rodents, but the extent to which these factors can influence the rewarding effects of oxycodone is unclear. The purpose of this study was to utilize place conditioning to determine the effects of sex and female hormonal status on the expression of oxycodone conditioned reward in rats. Gonadally intact adult Sprague-Dawley male and female rats were used to test: (1) whether both sexes express conditioned reward to oxycodone at similar doses, (2) the impact of conditioning session length on oxycodone conditioned reward expression in both sexes, and (3) the influence of female estrous cycle stage on oxycodone conditioned reward expression. Both sexes expressed conditioned reward at the same doses of oxycodone. Increasing the length of conditioning sessions did not reveal an effect of sex and resulted in lower magnitude conditioned reward expression. Importantly however, female stage of estrous cycle significantly influenced oxycodone conditioned reward expression. These results suggest that female hormonal status can impact the rewarding effects of opioids and thus have important implications for prescription opioid treatment practices.

Abstract Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.

Abstract. Background. Early neurologic deterioration (END) may occur in patients with anterior circulation ischemic stroke (ACIS) after receiving endovascular treatment (EVT). Hemodynamic insufficiency, re-occlusion, and post-re-canalization hyper-perfusion are likely to play a critical role in END. We hypothesized that hemodynamic changes can predict END in patients with ACIS post-successful EVT using trans-cranial Doppler (TCD). Methods. We utilized a prospectively maintained database of ACIS patients treated with EVT between September 2016 and June 2018 in the Xuanwu Hospital, Capital Medical University. TCD parameters including peak systolic velocity (PSV), bilateral mean flow velocity (MFV), and pulse index (PI) were determined via the middle cerebral arteries within 72 h post-EVT. A logistic regression model was applied to detect independent predictors for END. Results. Totally, 112 EVT patients were included in this study and 80/112 patients experienced successful re-canalization with

Background: Synthetic engineering of bacteria to produce industrial products is a burgeoning field of research and application. In order to optimize genome design, designers need to understand which genes are essential, which are optimal for growth, and locations in the genome that will be tolerated by the organism when inserting engineered cassettes. Methods: We present a pan-genome based method for the identification of core regions in a genome that are strongly conserved at the species level. Results: We show that the core regions determined by our method contain all or almost all essential genes. This demonstrates the accuracy of our method as essential genes should be core genes. We show that we outperform previous methods by this measure. We also explain why there are exceptions to this rule for our method. Conclusions: We assert that synthetic engineers should avoid deleting or inserting into these core regions unless they understand and are manipulating the function of the genes in that region. Similarly, if the designer wishes to streamline the genome, non-core regions and in particular low penetrance genes would be good targets for deletion. Care should be taken to remove entire cassettes with similar penetrance of the genes within cassettes as they may harbor toxin/antitoxin genes which need to be removed in tandem. The bioinformatic approach introduced here saves considerable time and effort relative to knockout studies on single isolates of a given species and captures a broad understanding of the conservation of genes that are core to a species.

Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria. Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in Bacillus subtilis ssp. subtilis. Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of Bacillus subtilis ssp. subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within B. subtilis or other bacterial species. Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.

Background: Synthetic engineering of bacteria to produce industrial products is a burgeoning field of research and application. In order to optimize genome design, designers need to understand which genes are essential, which are optimal for growth, and locations in the genome that will be tolerated by the organism when inserting engineered cassettes. Methods: We present a pan-genome based method for the identification of core regions in a genome that are strongly conserved at the species level. Results: We show that the core regions determined by our method contain all or almost all essential genes. This demonstrates the accuracy of our method as essential genes should be core genes. We show that we outperform previous methods by this measure. We also explain why there are exceptions to this rule for our method. Conclusions: We assert that synthetic engineers should avoid deleting or inserting into these core regions unless they understand and are manipulating the function of the genes in that region. Similarly, if the designer wishes to streamline the genome, non-core regions and in particular low penetrance genes would be good targets for deletion. Care should be taken to remove entire cassettes with similar penetrance of the genes within cassettes as they may harbor toxin/antitoxin genes which need to be removed in tandem. The bioinformatic approach introduced here saves considerable time and effort relative to knockout studies on single isolates of a given species and captures a broad understanding of the conservation of genes that are core to a species.

OBJECTIVES/GOALS: Several studies compare perioperative parameters and postoperative morphology of open cranial vault remodeling (CVR) and endoscopic-assisted craniectomy (EAC) in the treatment of sagittal craniosynostosis. However, data on neurodevelopmental outcomes comparing these techniques is lacking. METHODS/STUDY POPULATION: Patients with repaired sagittal synostosis were enrolled from a single tertiary care center between five and fifteen years of age. Patients with any craniofacial syndrome (e.g., 22q11.2 deletion) or other medical conditions that could affect neurodevelopment were excluded. Neurodevelopment was measured using the Differential Ability Scale-II (DAS-II). The General Cognitive Ability (GCA) score was the primary outcome measure, describing overall intellectual ability. Secondary outcome measures were measurements of cephalic index (CI) from pre- and postoperative imaging studies. Family socioeconomic status (SES) was estimated using the Hollingshead index (total score, continuous). RESULTS/ANTICIPATED RESULTS: 26 patients [10 CVR (2 Female:8 Male), 16 EAC (6 F:10 M)] were studied. Mean age at repair was greater with CVR than EAC (15.5 ± 8.5 months vs 3.5 ± 1.3 months, respectively, p < 0.001). Mean age at DAS-II testing was 9.0 ± 2.2 years. Patients GCA scores were within the average range relative to test norms (CVR 95.7 ± 15.4 vs EAC 102.6 ± 17.4). After adjusting for SES and preoperative CI, group differences in GCA were modest and statistically nonsignificant (p = 0.646). GCA scores were associated with SES (p = 0.054) but not preop CI (p = 0.479). Differences between CVR and EAC were not statistically significant for any of the imaging parameters (pre and postop CI, pre to postop change in CI, or age at postop imaging; 0.131

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain.

Objectives Omissions and delays in delivering nursing care are widely reported consequences of staffing shortages, with potentially serious impacts on patients. However, studies so far have relied almost exclusively on nurse self-reporting. Monitoring vital signs is a key part of nursing work and electronic recording provides an opportunity to objectively measure delays in care. This study aimed to determine the association between registered nurse (RN) and nursing assistant (NA) staffing levels and adherence to a vital signs monitoring protocol.Design Retrospective observational study.Setting 32 medical and surgical wards in an acute general hospital in England.Participants 538 238 nursing shifts taken over 30 982 ward days.Primary and secondary outcome measures Vital signs observations were scheduled according to a protocol based on the National Early Warning Score (NEWS). The primary outcome was the daily rate of missed vital signs (overdue by ≥67% of the expected time to next observation). The secondary outcome was the daily rate of late vital signs observations (overdue by ≥33%). We undertook subgroup analysis by stratifying observations into low, medium and high acuity using NEWS.Results Late and missed observations were frequent, particularly in high acuity patients (median=44%). Higher levels of RN staffing, measured in hours per patient per day (HPPD), were associated with a lower rate of missed observations in all (IRR 0.983, 95% CI 0.979 to 0.987) and high acuity patients (0.982, 95% CI 0.972 to 0.992). However, levels of NA staffing were only associated with the daily rate (0.954, CI 0.949 to 0.958) of all missed observations.Conclusions Adherence to vital signs monitoring protocols is sensitive to levels of nurse and NA staffing, although high acuity observations appeared unaffected by levels of NAs. We demonstrate that objectively measured omissions in care are related to nurse staffing levels, although the absolute effects are small.Study registration The data and analyses presented here were part of the larger Missed Care study (ISRCTN registration: 17930973).

OBJECTIVES/GOALS: The current opioid epidemic has placed post-operative pain management under scrutiny. Limiting post-operative pain can decrease overall opioid usage in the recovery period, especially after orthognathic surgery. Several studies have illustrated the efficacy of pregabalin in decreasing postoperative pain and opioid usage in adults undergoing orthognathic surgery. We aim to study the effects of a single dose of preoperative pregabalin on postoperative pain and total opioid consumption after orthognathic surgery in individuals with cleft lip and palate. METHODS/STUDY POPULATION: This was a retrospective cohort study of consecutive patients who received Le Fort I midface advancement between June 2012 and July 2019 by one of two surgeons at a single institution. We took advantage of our institution’s implementation, beginning in 2016, of a one-time dose of preoperative pregabalin for LeFort I midface advancement. All patients had diagnosed cleft lip and palate. The treatment group received a one-time preoperative dose of pregabalin. The control group did not receive pregabalin. Total morphine milligram equivalents (MME) consumption was calculated by adding intraoperative opioid administration and postoperative opioid consumption during admission. Postoperative pain control during admission consisted of oral oxycodone and intravenous (IV) hydromorphone or morphine. Duration of hospitalization and pain intensity assessed with the numeric pain rating scale (0-10) were also recorded. The mean postoperative pain assessment scores during admission was calculated for each patient. The median of these individual mean pain assessment scores for each group was subsequently computed. RESULTS/ANTICIPATED RESULTS: Twenty-three patients (14 males, 9 females) were included in this study; 12 patients received pregabalin (median dose: 150mg, range: 100-200mg). Mean age (years) at operation of the pregabalin (18.3 ± 1.9) and control groups (17.8 ± 1.9) were also equivalent (p = 0.571). Median hospital stay for both groups was 1.0 day. The pregabalin group had significantly lower consumption of total opioids during admission (total MME 70.95 MME; IQR: 24.65-150.17) compared to the control group (138.00 MME; IQR: 105.00-232.48) (MU = 31.00, p = 0.031). Although pain scores in the treatment group (3.21 ± 2.03) were lower than in the control group (3.71 ± 2.95), the difference was not statistically significant (p = 0.651, 95% Cl [−1.75, 2.75]). DISCUSSION/SIGNIFICANCE OF IMPACT: Based on the results, a one-time preoperative oral dose of pregabalin before orthognathic surgery in patients with cleft lip and palate reduced total opioid consumption during admission. However, there was no difference in length of stay or pain scores within the two groups. A single preemptive oral dose of pregabalin should be considered an effective adjunct to pain management protocols in patients undergoing orthognathic surgery.

The shape of a bypass graft plays an important role on its efficacy. Here, we investigated flow through two vascular graft designs-with and without cuff at the anastomosis. We conducted Digital Particle Image Velocimetry (DPIV) measurements to obtain the flow field information through these vascular grafts. Two pulsatile flow waveforms corresponding to cardiac cycles during the rest and the excitation states, with 10% and without retrograde flow out the proximal end of the native artery were examined. In the absence of retrograde flow, the straight end-to-side graft showed recirculation and stagnation regions that lasted throughout the full cardiac cycle with the stagnation region more pronounced in the excitation state. The contoured end-to-side graft had stagnation region that lasted only for a portion of the cardiac cycle and was less pronounced. With 10% retrograde flow, extended stagnation regions under both rest and excitation states for both bypass grafts were eliminated. Our results show that bypass graft designers need to consider both the type of flow waveform and presence of retrograde flow when sculpting an optimal bypass graft geometry.

The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 μM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

Multiple independent culture-based studies have identified the presence of Pseudomonas aeruginosa in respiratory samples as a positive risk factor for bronchiolitis obliterans syndrome (BOS). Yet, culture-independent microbiological techniques have identified a negative association between Pseudomonas species and BOS. Our objective was to investigate whether there may be a unifying explanation for these apparently dichotomous results.We performed bronchoscopies with bronchoalveolar lavage (BAL) on lung transplant recipients (46 procedures in 33 patients) and 26 non-transplant control subjects. We analyzed bacterial communities in the BAL fluid using qPCR and pyrosequencing of 16S rRNA gene amplicons and compared the culture-independent data with the clinical metadata and culture results from these subjects.Route of bronchoscopy (via nose or via mouth) was not associated with changes in BAL microbiota (p = 0.90). Among the subjects with positive Pseudomonas bacterial culture, P. aeruginosa was also identified by culture-independent methods. In contrast, a distinct Pseudomonas species, P. fluorescens, was often identified in asymptomatic transplant subjects by pyrosequencing but not detected via standard bacterial culture. The subject populations harboring these two distinct pseudomonads differed significantly with respect to associated symptoms, BAL neutrophilia, bacterial DNA burden and microbial diversity. Despite notable differences in culturability, a global database search of UM Hospital Clinical Microbiology Laboratory records indicated that P. fluorescens is commonly isolated from respiratory specimens.We have reported for the first time that two prominent and distinct Pseudomonas species (P. fluorescens and P. aeruginosa) exist within the post-transplant lung microbiome, each with unique genomic and microbiologic features and widely divergent clinical associations, including presence during acute infection.

The systemic inflammatory response to Clostridium difficile infection (CDI) is incompletely defined, particularly for patients with severe disease.Analysis of 315 blood samples from 78 inpatients with CDI (cases), 100 inpatients with diarrhea without CDI (inpatient controls), and 137 asymptomatic outpatient controls without CDI was performed. Serum or plasma was obtained from subjects at the time of CDI testing or shortly thereafter. Severe cases had intensive care unit admission, colectomy, or death due to CDI within 30 days after diagnosis. Thirty different circulating inflammatory mediators were quantified using an antibody-linked bead array. Principal component analysis (PCA), multivariate analysis of variance (MANOVA), and logistic regression were used for analysis.Based on MANOVA, cases had a significantly different inflammatory profile from outpatient controls but not from inpatient controls. In logistic regression, only chemokine (C-C motif) ligand 5 (CCL5) levels were associated with cases vs. inpatient controls. Several mediators were associated with cases vs. outpatient controls, especially hepatocyte growth factor, CCL5, and epithelial growth factor (inversely associated). Eight cases were severe and associated with elevations in IL-8, IL-6, and eotaxin.A broad systemic inflammatory response occurs during CDI and severe cases appear to differ from non-severe infections.

Clostridium difficile infection (CDI) is a major cause of morbidity and biomarkers that predict severity of illness are needed. Procalcitonin (PCT), a serum biomarker with specificity for bacterial infections, has been little studied in CDI. We hypothesized that PCT associated with CDI severity.Serum PCT levels were measured for 69 cases of CDI. Chart review was performed to evaluate the presence of severity markers and concurrent acute bacterial infection (CABI). We defined the binary variables clinical score as having fever (T >38°C), acute organ dysfunction (AOD), and/or WBC >15,000 cells/mm(3) and expanded score, which included the clinical score plus the following: ICU admission, no response to therapy, colectomy, and/or death.In univariate analysis log10 PCT associated with clinical score (OR 3.13, 95% CI 1.69-5.81, P0.2 ng/mL was 81% sensitive/73% specific for a positive clinical score and had a negative predictive value of 90%.An elevated PCT level associated with the presence of CDI severity markers and CDI was unlikely to be severe with a serum PCT level below 0.2 ng/mL. The extent to which PCT changes during CDI therapy or predicts recurrent CDI remains to be quantified.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.

BACKGROUND: We explored the use of routine blood tests and national early warning scores (NEWS) reported within ±24 hours of admission to predict in-hospital mortality in emergency admissions, using empirical decision Tree models because they are intuitive and may ultimately be used to support clinical decision making. METHODOLOGY: A retrospective analysis of adult emergency admissions to a large acute hospital during April 2009 to March 2010 in the West Midlands, England, with a full set of index blood tests results (albumin, creatinine, haemoglobin, potassium, sodium, urea, white cell count and an index NEWS undertaken within ±24 hours of admission). We developed a Tree model by randomly splitting the admissions into a training (50%) and validation dataset (50%) and assessed its accuracy using the concordance (c-) statistic. Emergency admissions (about 30%) did not have a full set of index blood tests and/or NEWS and so were not included in our analysis. RESULTS: There were 23248 emergency admissions with a full set of blood tests and NEWS with an in-hospital mortality of 5.69%. The Tree model identified age, NEWS, albumin, sodium, white cell count and urea as significant (p

The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (Brg1(null/+)) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1 floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that Brg1(Wap-Cre) conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1(Wap-Cre) mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined to particular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations in human ovarian and endometrial tumors.

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca(2+) through L-type voltage gated Ca(2+) channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca(2+)](i) or Ca(2+) efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca(2+)](i) induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca(2+) chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca(2+) signalling to ERK, we provide evidence that a sustained increase in Ca(2+) within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrients has many physiological effects but particularly enhances glucose-dependent insulin release through the GLP-1 receptor (GLP-1R). GLP-1 7-36 amide, the predominant circulating active form of GLP-1, is rapidly truncated by dipeptidyl peptidase-4 to GLP-1 9-36 amide, which is generally considered inactive. Given its physiological roles, the GLP-1R is targeted for treatment of type 2 diabetes. Recently 'compound 2' has been described as both an agonist and positive allosteric modulator of GLP-1 7-36 amide affinity, but not potency, at the GLP-1R. Importantly, we demonstrated previously that exendin 9-39, generally considered a GLP-1R antagonist, enhances compound 2 efficacy (or vice versa) at the GLP-1R. Given that GLP-1 9-36 amide is the major circulating form of GLP-1 post-prandially and is a low affinity weak partial agonist or antagonist at the GLP-1R, we investigated interaction between this metabolite and compound 2 in a cell line with recombinant expression of the human GLP-1R and the rat insulinoma cell line, INS-1E, with native expression of the GLP-1R. We show compound 2 markedly enhances efficacy and potency of GLP-1 9-36 amide for key cellular responses including AMP generation, Ca(2+) signaling and extracellular signal-regulated kinase. Thus, metabolites of peptide hormones including GLP-1 that are often considered inactive may provide a means of manipulating key aspects of receptor function and a novel therapeutic strategy.

Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry ("healthy smokers", HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Sensitization to the effects of drugs of abuse and associated stimuli contributes to drug craving, compulsive drug use, and relapse in addiction. Repeated opiate exposure produces behavioral sensitization that is hypothesized to result from neural plasticity in specific limbic, striatal and cortical systems. ΔFosB and FosB are members of the Fos family of transcription factors that are implicated in neural plasticity in addiction. This study examined the effects of intermittent morphine treatment, associated with motor sensitization, on FosB/ΔFosB levels using quantitative immunohistochemistry. Motor sensitization was tested in C57BL/6 mice that received six intermittent pre-treatments (on days 1, 3, 5, 8, 10, 12) with either subcutaneous morphine (10 mg/kg) or saline followed by a challenge injection of morphine or saline on day 16. Mice receiving repeated morphine injections demonstrated significant increases in locomotor activity on days 8, 10, and 12 of treatment (vs. day 1), consistent with development of locomotor sensitization. A morphine challenge on day 16 significantly increased locomotor activity of saline pre-treated mice and produced even larger increases in motor activity in the morphine pre-treated mice, consistent with the expression of opiate sensitization. Intermittent morphine pre-treatment on these six pre-treatment days produced a significant induction of FosB/ΔFosB, measured on day 16, in multiple brain regions including prelimbic (PL) and infralimbic (IL) cortex, nucleus accumbens (NAc) core, dorsomedial caudate-putamen (CPU), basolateral amygdala (BLA) and central nucleus of the amygdala (CNA) but not in a motor cortex control region. Opiate induced sensitization may develop via Fos/ΔFosB plasticity in motivational pathways (NAc), motor outputs (CPU), and associative learning (PL, IL, BLA) and stress pathways (CNA).

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription) complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II) passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16--the Spt16-M domain--is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.

There is evidence that immune-activated macrophages infected with the Human Immunodeficiency Virus (HIV) are associated with tissue damage and serve as a long-lived viral reservoir during therapy. In this study, we analyzed 780 HIV genetic sequences generated from 53 tissues displaying normal and abnormal histopathology. We found up to 50% of the sequences from abnormal lymphoid and macrophage rich non-lymphoid tissues were intra-host viral recombinants. The presence of extensive recombination, especially in non-lymphoid tissues, implies that HIV-1 infected macrophages may significantly contribute to the generation of elusive viral genotypes in vivo. Because recombination has been implicated in immune evasion, the acquisition of drug-resistance mutations, and alterations of viral co-receptor usage, any attempt towards the successful eradication of HIV-1 requires therapeutic approaches targeting tissue macrophages.

Abstract The Arctic is experiencing rapid reductions in sea ice, affecting all ice-dependant species. In the present study we examine interannual seasonal movements and habitat use in relation to sea ice coverage for one of the Arctic endemic marine mammals. We tagged 40 male walruses (Odobenus rosmarus) in the Svalbard Archipelago with custom-designed tusk-mounted GPS loggers. Twelve of these animals provided tracks that lasted 1–6 years. Eleven of the walruses displayed clear seasonal migratory behaviour between summer foraging areas and winter breeding areas. Individuals showed high inter-individual variation, but clear site fidelity, using the same areas in consecutive years despite variable sea ice conditions. The walruses swam 5225–10,406 km per year and travelled remarkably similar distances between years on an individual basis. The phenology of migration was not impacted by sea ice concentrations or daylight length but was consistent at the individual level, suggesting endogenous drivers. Sea ice concentrations influenced movement behaviour with animals showing more tortuous paths when in areas with heavy sea ice, possibly searching for polynyas where females reside. Ongoing climate change is expected to drastically change walrus habitat, and it remains to be seen if walruses will be able to shift from their fixed seasonal migratory routines.