Melanin-Concentrating Hormone Receptor-1 is Enriched in Lipid Rafts and the Effects of Lipid Raft Integrity on Receptor Signaling

The melanin-concentrating hormone receptor-1 (MCHR-1) is a member of the G protein-coupled receptor (GPCR) superfamily, and functions in the regulation of food consumption and energy metabolism. MCHR-1 is expressed in neural, pancreatic, and fat tissue. Fat cells begin their journey as pre-adipocytes, culminating in the formation of mature adipocytes as differentiation occurs. Of interest to note, fat cells accumulate caveolae markers -caveolin-1 and cholesterol -during this process. Certain GPCRs and their associated downstream signaling molecules co-localize with caveolae membranes. This thesis seeks to demonstrate that MCHR-1 is enriched in lipid rafts and that a possible consequence of this may be altered signal transduction in obese individuals, such as downregulated ERK 1/2-mediated leptin transcription. Increased amounts of adipose tissue, and thus caveolae, may play a central role in the regulation of MCHR-1 signaling. The first aim of this project addressed the question of MCHR-1 localization to lipid rafts. To test this, caveolae membranes were isolated via sucrose density gradient ultracentrifugation. Subsequent fractionation and western blotting confirmed that MCHR-1 is enriched in lipid raft fractions containing caveolin-1. The second objective assessed ligand dependence on MCHR-1 localization. MCH exposure (1.0-µM) had no obvious effect on receptor localization to lipid raft fractions containing caveolin-1 over an expanded time course. The third aim examined the effect of lipid rafts on MCHR-1 signaling. Pharmacological disruption of caveolae by cholesterol depletion with methyl­ β-cyclodextrin (MβCD) dampened MCH-mediated ERK 1/2 activation, suggesting that lipid rafts may significantly impact the regulation of MCHR-1 signaling in cells

Virus-Host Interactions at the Maternal-Fetal Interface

Strategies to protect against viral infections are essential during pregnancy. Maternal-fetal transmission can have serious pathological outcomes, including fetal infection, growth restriction, birth defects, and/or death. Throughout pregnancy, the placenta (composed of polarized trophoblasts amid stromal and vascular arrangements) is an indispensable tissue that forms a barrier at the maternal-fetal interface. Viruses have likely evolved specific mechanisms to exploit the protective functions of placental trophoblasts to initiate fetal infection. Despite the severity of pathologic disease associated with fetal viral infection, little is known regarding virus-host interactions at the maternal-fetal interface. In this work, we have examined the mechanisms by which – 1) placental trophoblasts protect against invading viruses and 2) coxsackievirus B (CVB), a virus associated with fetal pathology, gains entry into polarized trophoblasts. As a model, we have used cultured primary human trophoblasts (PHTs) and immortalized human (BeWo) trophoblasts. We have found that PHTs are highly resistant to infection by six disparate viruses. PHTs transfer this resistance to non-placental recipient cells through exosome-mediated delivery of select placental microRNAs (miRNAs). We show that members of the chromosome 19 miRNA cluster (C19MC), which are almost exclusively expressed in the primate placenta, are packaged within trophoblast-derived exosomes, and attenuate viral replication in recipient cells by inducing autophagy. To study CVB entry into placental trophoblasts, we have merged virological and cell biological techniques, combined with pharmacological inhibitors and siRNAs directed against diverse cellular endocytic and signaling components, to characterize the pathways hijacked by CVB to promote its entry into human trophoblasts. We found the kinetics of CVB entry and uncoating in placental trophoblasts similar to those described in polarized intestinal epithelial cells. CVB entry into placental trophoblasts requires decay accelerating factor (DAF) binding, and is associated with the relocalization of virus from the apical surface to intercellular tight junctions. We have identified a divergent mechanism for CVB entry that is independent of clathrin, caveolae, and dynamin II but is dependent on lipid-rafts and Src family tyrosine kinase signaling. Our studies model viral transmission and infection at the maternal-fetal interface, and have the therapeutic potential for preventing prenatal infections, pre-term labor, and birth defects

On the edge of degradation: Autophagy regulation by RNA decay

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149292/1/wrna1522_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149292/2/wrna1522.pd

Роль средств мультимедиа в современной хореографии

Цель исследования данной статьи - рассмотреть особенности взаимовлияния мультимедийных технологий и хореографии конца ХХ начала XXI веков

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition

A three-dimensional culture system recapitulates placental syncytiotrophoblast development and microbial resistance

In eutherians, the placenta acts as a barrier and conduit at the maternal-fetal interface. Syncytiotrophoblasts, the multinucleated cells that cover the placental villous tree surfaces of the human placenta, are directly bathed in maternal blood and are formed by the fusion of progenitor cytotrophoblasts that underlie them. Despite their crucial role in fetal protection, many of the events that govern trophoblast fusion and protection from microbial infection are unknown. We describe a three-dimensional (3D)–based culture model using human JEG-3 trophoblast cells that develop syncytiotrophoblast phenotypes when cocultured with human microvascular endothelial cells. JEG-3 cells cultured in this system exhibit enhanced fusogenic activity and morphological and secretory activities strikingly similar to those of primary human syncytiotrophoblasts. RNASeq analyses extend the observed functional similarities to the transcriptome, where we observed significant overlap between syncytiotrophoblast-specific genes and 3D JEG-3 cultures. Furthermore, JEG-3 cells cultured in 3D are resistant to infection by viruses and Toxoplasma gondii, which mimics the high resistance of syncytiotrophoblasts to microbial infections in vivo. Given that this system is genetically manipulatable, it provides a new platform to dissect the mechanisms involved in syncytiotrophoblast development and microbial resistance

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.Peer reviewe

Agriculture and food systems in sub-Saharan Africa in a 4 degrees C+ world

Agricultural development in sub-Saharan Africa faces daunting challenges, which climate change and increasing climate variability will compound in vulnerable areas. The impacts of a changing climate on agricultural production in a world that warms by 4°C or more are likely to be severe in places. The livelihoods of many croppers and livestock keepers in Africa are associated with diversity of options. The changes in crop and livestock production that are likely to result in a 4°C+ world will diminish the options available to most smallholders. In such a world, current crop and livestock varieties and agricultural practices will often be inadequate, and food security will be more difficult to achieve because of commodity price increases and local production shortfalls. While adaptation strategies exist, considerable institutional and policy support will be needed to implement them successfully on the scale required. Even in the 2°C+ world that appears inevitable, planning for and implementing successful adaptation strategies are critical if agricultural growth in the region is to occur, food security be achieved and household livelihoods be enhanced. As part of this effort, better understanding of the critical thresholds in global and African food systems requires urgent research

Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches