A simple method to identify kinases that regulate embryonic stem cell pluripotency by high-throughput inhibitor screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed "naïve" and "primed" pluripotency. The mechanisms that control naïve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify naïve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds

Audit-identified avoidable factors in maternal and perinatal deaths in low resource settings: a systematic review

Background: Audits provide a rational framework for quality improvement by systematically assessing clinical practices against accepted standards with the aim to develop recommendations and interventions that target modifiable deficiencies in care. Most childbirth-associated mortality audits in developing countries are focused on a single facility and, up to now, the avoidable factors in maternal and perinatal deaths cataloged in these reports have not been pooled and analyzed. We sought to identity the most frequent avoidable factors in childbirth-related deaths globally through a systematic review of all published mortality audits in low and lower-middle income countries. Methods: We performed a systematic review of published literature from 1965 to November 2011 in Pubmed, Embase, CINAHL, POPLINE, LILACS and African Index Medicus. Inclusion criteria were audits from low and lower-middle income countries that identified at least one avoidable factor in maternal or perinatal mortality. Each study included in the analysis was assigned a quality score using a previously published instrument. A meta-analysis was performed for each avoidable factor taking into account the sample sizes and quality score from each individual audit. The study was conducted and reported according to PRISMA guidelines for systematic reviews. Results: Thirty-nine studies comprising 44 datasets and a total of 6,205 audited deaths met inclusion criteria. The analysis yielded 42 different avoidable factors, which fell into four categories: health worker-oriented factors, patient-oriented factors, transport/referral factors, and administrative/supply factors. The top three factors by attributable deaths were substandard care by a health worker, patient delay, and deficiencies in blood transfusion capacity (accounting for 688, 665, and 634 deaths attributable, respectively). Health worker-oriented factors accounted for two-thirds of the avoidable factors identified. Conclusions: Audits provide insight into where systematic deficiencies in clinical care occur and can therefore provide crucial direction for the targeting of interventions to mitigate or eliminate health system failures. Given that the main causes of maternal and perinatal deaths are generally consistent across low resource settings, the specific avoidable factors identified in this review can help to inform the rational design of health systems with the aim of achieving continued progress towards Millennium Development Goals Four and Five. Electronic supplementary material The online version of this article (doi:10.1186/1471-2393-14-280) contains supplementary material, which is available to authorized users

Disputed Churches in Jakarta

Healthy pluralism requires space for all religious adherents to worship and construct places of worship in accordance with their convictions. The state should protect this right as an essential matter. Despite this normative ideal, there is still much controversy surrounding the construction of places of worship in Indonesia. In the last few years, the planned construction of a number of places of worship has been disputed, although others have been able to overcome these problems by relying on different strategies. This research seeks to examine the factors that play a role in initiating and resolving conflict over places of worship. Places of worship are specifically limited in this study to Catholic churches and Protestant churches that are members of the Communion of Churches in Indonesia (Persekutuan Gereja-gereja di Indonesia, PGI). The methods used were participant observation and in-depth interviews of churches representing one of four categories: (1) undisputed churches; (2) disputed churches that have since resolved the dispute; (3) originally undisputed churches that have since become disputed; and (4) churches that have never been able to resolve the dispute. Based on thirteen case studies, the research on which this report is based confirms the influential role of state regulation and social factors. The cases show that the obstacles some churches experience are generally related to weak government agencies due to political, social or ideological reasons. In terms of social factors, demographic factors were not found to have an influence. Resistance to churches was more often caused by a lack of communication, or provocation, or intimidation by specific groups. After describing and analysing the thirteen cases selected, this monograph closes with conclusions and recommendations

Cell-specific discrimination of desmosterol and desmosterol mimetics confers selective regulation of LXR and SREBP in macrophages.

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia

An integrated biochemical prediction model of all-cause mortality in patients undergoing lower extremity bypass surgery for advanced peripheral artery disease

BackgroundPatients with advanced peripheral artery disease (PAD) have a high prevalence of cardiovascular (CV) risk factors and shortened life expectancy. However, CV risk factors poorly predict midterm (<5 years) mortality in this population. This study tested the hypothesis that baseline biochemical parameters would add clinically meaningful predictive information in patients undergoing lower extremity bypass operations.MethodsThis was a prospective cohort study of patients with clinically advanced PAD undergoing lower extremity bypass surgery. The Cox proportional hazard model was used to assess the main outcome of all-cause mortality. A clinical model was constructed with known CV risk factors, and the incremental value of the addition of clinical chemistry, lipid assessment, and a panel of 11 inflammatory parameters was investigated using the C statistic, the integrated discrimination improvement index, and Akaike information criterion.ResultsThe study monitored 225 patients for a median of 893 days (interquartile range, 539-1315 days). In this study, 50 patients (22.22%) died during the follow-up period. By life-table analysis (expressed as percent surviving ± standard error), survival at 1, 2, 3, 4, and 5 years, respectively, was 90.5% ± 1.9%, 83.4% ± 2.5%, 77.5% ± 3.1%, 71.0% ± 3.8%, and 65.3% ± 6.5%. Compared with survivors, decedents were older, diabetic, had extant coronary artery disease, and were more likely to present with critical limb ischemia as their indication for bypass surgery (P < .05). After adjustment for the above, clinical chemistry and inflammatory parameters significant (hazard ratio [95% confidence interval]) for all-cause mortality were albumin (0.43 [0.26-0.71]; P = .001), estimated glomerular filtration rate (0.98 [0.97-0.99]; P = .023), high-sensitivity C-reactive protein (hsCRP; 3.21 [1.21-8.55]; P = .019), and soluble vascular cell adhesion molecule (1.74 [1.04-2.91]; P = .034). Of the inflammatory molecules investigated, hsCRP proved most robust and representative of the integrated inflammatory response. Albumin, eGFR, and hsCRP improved the C statistic and integrated discrimination improvement index beyond that of the clinical model and produced a final C statistic of 0.82.ConclusionsA risk prediction model including traditional risk factors and parameters of inflammation, renal function, and nutrition had excellent discriminatory ability in predicting all-cause mortality in patients with clinically advanced PAD undergoing bypass surgery

DEPTOR Is an mTOR Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival.Howard Hughes Medical Institute (Investigator)Dana-Farber Cancer Institute (High-Tech Research Fund)National Cancer Institute (U.S.)National Institutes of Health (U.S.) (Intramural Research Program)American Cancer SocietyCanadian Institutes of Health Research (Fellowship)American Diabetes Association (Fellowship)W. M. Keck FoundationNational Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (NIH; R01 AI47389

A single center phase II study of ixazomib in patients with relapsed or refractory cutaneous or peripheral T‐cell lymphomas

The transcription factor GATA‐3, highly expressed in many cutaneous T‐cell lymphoma (CTCL) and peripheral T‐cell lymphomas (PTCL), confers resistance to chemotherapy in a cell‐autonomous manner. As GATA‐3 is transcriptionally regulated by NF‐κB, we sought to determine the extent to which proteasomal inhibition impairs NF‐κB activation and GATA‐3 expression and cell viability in malignant T cells. Proteasome inhibition, NF‐κB activity, GATA‐3 expression, and cell viability were examined in patient‐derived cell lines and primary T‐cell lymphoma specimens ex vivo treated with the oral proteasome inhibitor ixazomib. Significant reductions in cell viability, NF‐κB activation, and GATA‐3 expression were observed preclinically in ixazomib‐treated cells. Therefore, an investigator‐initiated, single‐center, phase II study with this agent in patients with relapsed/refractory CTCL/PTCL was conducted. Concordant with our preclinical observations, a significant reduction in NF‐κB activation and GATA‐3 expression was observed in an exceptional responder following one month of treatment with ixazomib. While ixazomib had limited activity in this small and heterogeneous cohort of patients, inhibition of the NF‐κB/GATA‐3 axis in a single exceptional responder suggests that ixazomib may have utility in appropriately selected patients or in combination with other agents.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139920/1/ajh24895.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139920/2/ajh24895_am.pd

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors.

The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection