Herpes B Virus,Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8⁺ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions.     

Structure prediction and binding sites analysis of curcin protein of Jatropha curcas using computational approaches

Ribosome inactivating proteins (RIPs) are defense proteins in a number of higher-plant species that are directly targeted toward herbivores. Jatropha curcas is one of the biodiesel plants having RIPs. The Jatropha seed meal, after extraction of oil, is rich in curcin, a highly toxic RIP similar to ricin, which makes it unsuitable for animal feed. Although the toxicity of curcin is well documented in the literature, the detailed toxic properties and the 3D structure of curcin has not been determined by X-ray crystallography, NMR spectroscopy or any in silico techniques to date. In this pursuit, the structure of curcin was modeled by a composite approach of 3D structure prediction using threading and ab initio modeling. Assessment of model quality was assessed by methods which include Ramachandran plot analysis and Qmean score estimation. Further, we applied the protein-ligand docking approach to identify the r-RNA binding residue of curcin. The present work provides the first structural insight into the binding mode of r-RNA adenine to the curcin protein and forms the basis for designing future inhibitors of curcin. Cloning of a future peptide inhibitor within J. curcas can produce non-toxic varieties of J. curcas, which would make the seed-cake suitable as animal feed without curcin detoxification.     

Placenta Stem/Stromal Cell–Derived Extracellular Vesicles for Potential Use in Lung Repair

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next‐generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue‐repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.     

Quaternary variations in the structural assembly of N‐acetylglucosamine‐6‐phosphate deacetylase from Pasteurella multocida

N‐acetylglucosamine 6‐phosphate deacetylase (NagA) catalyzes the conversion of N‐acetylglucosamine‐6‐phosphate to glucosamine‐6‐phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo‐microscopy. Analysis of the determined crystal structure reveals a set of hot‐spot residues involved in novel interactions at the dimer‐dimer interface. PmNagA binds to one Zn²⁺ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10‐fold the K_M), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.     

Rapid encoding of an internal model for imitative learning

As in human infant speech development, vocal imitation in songbirds involves sensory acquisition and memorization of adult-produced vocal signals, followed by a protracted phase of vocal motor practice. The internal model of adult tutor song in the juvenile male brain, termed ‘the template’, is central to the vocal imitation process. However, even the most fundamental aspects of the template, such as when, where and how it is encoded in the brain, remain poorly understood. A major impediment to progress is that current studies of songbird vocal learning use protracted tutoring over days, weeks or months, complicating dissection of the template encoding process. Here, we take the key step of tightly constraining the timing of template acquisition. We show that, in the zebra finch, template encoding can be time locked to, on average, a 2 h period of juvenile life and based on just 75 s of cumulative tutor song exposure. Crucially, we find that vocal changes occurring on the day of training correlate with eventual imitative success. This paradigm will lead to insights on how the template is instantiated in the songbird brain, with general implications for deciphering how internal models are formed to guide learning of complex social behaviours.     

Staurosporine-induced Growth Inhibition of Glioma Cells is Accompanied by Altered Expression of Cyclins, CDKs and CDK Inhibitors

Staurosporine was found to bring about complete growth inhibition of human glioma cell lines. U87 MG cells were arrested in S phase while U373 MG cells in G2/M phase on staurosporine treatment. Consistent with this observation, no change in G1 phase regulators viz., Cyclin D1, D3 and CDK4 was seen on staurosporine treatment. The levels of CDK2, CDC2, Cyclin A and Cyclin B proteins decreased, while the levels of CDK inhibitors viz., p21 and p27 were found to increase on staurosporine treatment. The mRNA levels of CDK2 and CDC2 genes were also found to decrease on staurosporine treatment. Thus apart from staurosporine’s known direct inhibitory effect on CDK2 and CDC2 activities, staurosporine was found to down-regulate activities of these two kinases by modulating the expression of the kinases themselves as well that of their activating partners (Cyclins) and their inhibitors.     

A Population Proportion approach for ranking differentially expressed genes

Background  

DNA microarrays are used to investigate differences in gene expression between two or more classes of samples. Most currently used approaches compare mean expression levels between classes and are not geared to find genes whose expression is significantly different in only a subset of samples in a class. However, biological variability can lead to situations where key genes are differentially expressed in only a subset of samples. To facilitate the identification of such genes, a new method is reported.     

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.