Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

Metabolites from intestinal microbes shape Treg

Intestinal bacterial metabolites are an important communication tool between the host immune system and the commensal microbiota to establish mutualism. In a recent paper published in Science, Wendy Garrett and her colleagues report an exciting role of the three most abundant microbial-derived short-chain fatty acids (SCFA), acetic acid, propionic acid and butyric acid, in colonic regulatory T cell (cTreg) homeostasis.A number of studies have shown that increased cTreg numbers and their immunoregulatory function are promoted by the presence of commensal intestinal microbes (either individual species such as Bacteroides fragilis¹, defined benign consortia of bacteria such as the altered Schaedler flora² or groups of Clostridia³). In a recent paper in Science, Garrett and colleagues report how these effects are generated through molecular exchanges between the host and the enormous load of microbes carried in the lower intestine⁴.Smith et al.⁴ investigated the role of SCFA, which are bacterial fermentation products produced by a wide variety of bacteria through anaerobic acidogenic pathways. SCFA released by colonic bacteria have long been known to be important as a carbon source for colonic epithelial cells⁵. From this new work we can now see that signaling effects of SCFA also regulate cTreg homeostasis.Microbiota-derived SCFA were found to increase total (thymic-derived) cTreg numbers. The homing characteristics to the colon and the regulatory functions of these cells (such as IL-10 production) were also enhanced through SCFA treatment.These effects are mediated by the G-protein-coupled free fatty acid receptor 43 (GPR43). Using mice that are genetically deficient in this receptor, Smith et al. showed that this signaling pathway is responsible for the increased cTreg numbers in vivo and that signaling by SCFA reduces the susceptibility to chronic intestinal inflammation. As they found GPR43 expression on cTreg (compared with lower GPR43 expression on Treg from other sites) this may be a direct effect, e.g. alterations in histone deacetylation. However, other cell types in the GI tract also express GPR43, including enteroendocrine cells and other leukocytes, therefore indirect effects are not yet excluded. In fact, Atarashi and colleagues have recently published their studies of how Clostridial species induce cTreg⁶. They found that bacterial-derived SCFA stimulate epithelial cells to produce TGFβ, contributing to Treg differentiation and expansion.Whereas other species-specific bacterial molecules, such as B. fragilis-derived PSA, have previously been demonstrated to have immunomodulatory functions², the report by Smith et al. is an elegant demonstration of the ubiquitous and pervasive bacterial metabolites that impact on the mucosal immune system. There is really a rather promiscuous exchange of metabolites between the microbiota and the host, with metabolic pathways that require components of both eukaryotic and prokaryotic cells. Bile acids are a great example of such a mixed pathway, where a dysbiosis caused by obesity promotes liver cancer through alterations in the microbial bile acid metabolism⁷. Although Smith et al. do not see any SCFA-mediated effects on central Treg compartments (outside the colon), other bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed increased incidence of intestinal inflammation and systemic immune-mediated disorders such as autoimmune or allergic diseases (Figure 1), which are often linked to changes within the microbiota due to diet or antibiotic use⁸.Open in a separate windowFigure 1Bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed correlation of microbiota composition and susceptibility to systemic immune-mediated disorders such as autoimmune or allergic diseases.A clinical situation in which the colon faces a deficiency of SCFA happens after surgery that diverts the fecal stream into a stoma bag, leaving the distal colon without its normal contents. This operation may be carried out to protect a low surgical anastomosis after removal of a tumor. The result is that the defunctioned colon frequently becomes inflamed, a condition recognized as ''diversion colitis''. In some cases, treatment with SCFA has been able to treat the condition successfully⁹. The lack of SCFA as a carbon source for colonocytes was previously considered as a key factor in the aetiopathogenesis of the condition, although this will need to be reviewed in the light of the new data on the effects of SFCA on colonic Treg numbers and function.Our colonic health depends on our intestinal microbiota and what we feed them. Changes in Western dietary patterns, e.g., due to reduced intake of plant fibers, might drastically impact the production of SCFA within the intestine. Furthermore, Smith et al. demonstrate a direct effect of antibiotic (vancomycin) treatment on SCFA levels, which in turn affects intestinal immune regulation by reducing the number of cTreg.Taken together, this draws a picture of a superorganism composed of the host (us) and our microbiota, with the metabolic interface as an important communication tool. This allows the host and the microbiota to adapt to and communicate with each other. Originally, germ-free animals were derived to challenge the notion that the existence of higher organisms was irrevocably linked to their associated microbiotas¹⁰. Although the germ-free program succeeded¹¹, it has provided us with powerful tools to show that the original notion was justified: pervasive metabolic interactions and signaling make us the sum of our prokaryotic and eukaryotic cellular components.     

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle,independently of its on-target effect

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.     

Chimpanzees predict that a competitor's preference will match their own

The ability to predict how another individual will behave is useful in social competition. Chimpanzees can predict the behaviour of another based on what they observe her to see, hear, know and infer. Here we show that chimpanzees act on the assumption that others have preferences that match their own. All subjects began with a preference for a box with a picture of food over one with a picture of nothing, even though the pictures had no causal relation to the contents. In a back-and-forth food competition, chimpanzees then avoided the box with the picture of food when their competitor had chosen one of the boxes before them—presumably on the assumption that the competitor shared their own preference for it and had already chosen it. Chimpanzees predicted that their competitor''s preference would match their own and adjusted their behavioural strategies accordingly.     

Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode,Rotylenchulus reniformis

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.     

Barn owls (<Emphasis Type="Italic">Tyto alba</Emphasis>) in western North America: phylogeographic structure,connectivity, and genetic diversity

The barn owl (Tyto alba) is a non-migratory species widely distributed across much of North America in areas with extensive old-field and grassland habitat and without extensive winter snow cover. We investigated the genetic diversity and phylogeographic patterns of barn owl populations in western North America, ranging from British Columbia (BC) to southern California, and one eastern population from Pennsylvania. We also determined the genetic distinctiveness of a population off the coast of southern California, Santa Barbara Island, as management plans to control the local owl population are being considered to decrease predation rate on the now threatened Scripps’s Murrelet (Synthliboramphus scrippsi). Using 8 polymorphic microsatellite markers (N = 126) and ND2 mitochondrial sequences (N = 37), we found little to no genetic structure among all sampled regions, with the exception of Santa Barbara Island. The BC mainland population, despite its northwestern geographically peripheral location and ongoing habitat degradation, is not genetically depauperate. However, individuals from Vancouver Island, likewise a peripheral population in BC, exhibited the lowest genetic diversity of all sampled locations. The low global F_ST value (0.028) estimated from our study suggests that old-field agricultural habitats are well connected in North America. Since the BC population has declined by about 50 % within the last three decades, it is vital to focus on preserving the remaining barn owl habitats in BC to allow successful establishment from neighbouring populations. Additionally, our microsatellite data revealed that the population on Santa Barbara Island showed genetic divergence from its continental counterpart. Mitochondrial data, however, demonstrated that this island population is not a monophyletic lineage containing unique haplotypes, and hence cannot be designated as an Evolutionarily Significant Unit.     

MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC₅₀s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.     

Long-Lasting Immune Protection and Other Epidemiological Findings after Chikungunya Emergence in a Cambodian Rural Community,April 2012

The East/Central/South African genotype of Chikungunya virus with the E1-A226V mutation emerged in 2011 in Cambodia and spread in 2012. An outbreak of 190 cases was documented in Trapeang Roka, a rural village. We surveyed 425 village residents within 3–4 weeks after the outbreak, and determined the sensitivity and specificity of case definitions and factors associated with infection by CHIKV. Self-reported clinical presentation consisted mostly of fever, rash and arthralgia. The presence of all three clinical signs or symptoms was identified as the most sensitive (67%) and specific (84%) self-reported diagnostic clinical indicator compared to biological confirmation by MAC-ELISA or RT-PCR used as a reference. Having an indoor occupation was associated with lower odds of infection compared with people who remained at home (adjOR 0.32, 95%CI 0.12–0.82). In contrast with findings from outbreaks in other settings, persons aged above 40 years were less at risk of CHIKV infection, likely reflecting immune protection acquired when Chikungunya circulated in Cambodia before the Khmer Rouge regime in 1975. In view of the very particular history of Cambodia, our epidemiological data from Trapeang Roka are the first to support the persistence of CHIKV antibodies over a period of 40 years.     

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation

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