Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Software system to predict the infection in COVID-19 patients using deep learning and web of things.

Since the end of 2019, computed tomography (CT) images have been used as an important substitute for the time-consuming Reverse Transcriptase polymerase chain reaction (RT-PCR) test; a new coronavirus 2019 (COVID-19) disease has been detected and has quickly spread through many countries across the world. Medical imaging such as computed tomography provides great potential due to growing skepticism toward the sensitivity of RT-PCR as a screening tool. For this purpose, automated image segmentation is highly desired for a clinical decision aid and disease monitoring. However, there is limited publicly accessible COVID-19 image knowledge, leading to the overfitting of conventional approaches. To address this issue, the present paper focuses on data augmentation techniques to create synthetic data. Further, a framework has been proposed using WoT and traditional U-Net with EfficientNet B0 to segment the COVID Radiopedia and Medseg datasets automatically. The framework achieves an F-score of 0.96, which is best among state-of-the-art methods. The performance of the proposed framework also computed using Sensitivity, Specificity, and Dice-coefficient, achieves 84.5%, 93.9%, and 65.0%, respectively. Finally, the proposed work is validated using three quality of service (QoS) parameters such as server latency, response time, and network latency which improves the performance by 8%, 7%, and 10%, respectively.

A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation.

Traditional functional genetic studies in crops are time consuming, complicated and cannot be readily scaled up. The reason is that mutant or transformed crops need to be generated to study the effect of gene modifications on specific traits of interest. However, many crop species have a complex genome and a long generation time. As a result, it usually takes several months to over a year to obtain desired mutants or transgenic plants, which represents a significant bottleneck in the development of new crop varieties. To overcome this major issue, we are currently establishing a versatile plant genetic screening platform, amenable to high throughput screening in almost any crop species, with a unique workflow. This platform combines protoplast transformation and fluorescence activated cell sorting. Here we show that tobacco protoplasts can accumulate high levels of lipid if transiently transformed with genes involved in lipid biosynthesis and can be sorted based on lipid content. Hence, protoplasts can be used as a predictive tool for plant lipid engineering. Using this newly established strategy, we demonstrate the major role of ABI3 in plant lipid accumulation. We anticipate that this workflow can be applied to numerous highly valuable metabolic traits other than storage lipid accumulation. This new strategy represents a significant step toward screening complex genetic libraries, in a single experiment and in a matter of days, as opposed to years by conventional means.

Class-Switch Recombination Occurs Infrequently in Germinal Centers.

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.

TFH-derived dopamine accelerates productive synapses in germinal centres.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

Deriving and testing of dysplastic murine hepatocytes: A new platform in liver cancer research.

Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of "hepatoma" cell lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemistry. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G2/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesis.

The effect of alpha-lipoic acid on mitochondrial superoxide and glucocorticoid-induced hypertension.

AIMS: To examine the effect of alpha-lipoic acid, an antioxidant with mitochondrial superoxide inhibitory properties, on adrenocorticotrophic hormone- (ACTH-HT) and dexamethasone-induced hypertensions (DEX-HT) in rats and if any antihypertensive effect is mediated via mitochondrial superoxide inhibition. METHODS: In a prevention study, rats received ground food or alpha-lipoic-acid-laced food (10 mg/rat/day) for 15 nights. Saline, adrenocorticotrophic hormone (ACTH, 0.2 mg/kg/day), or dexamethasone (DEX, 10 µ g/rat/day) was injected subcutaneously from day 5 to day 11. In a reversal study, rats received alpha-lipoic-acid-laced food 4 days after commencement of saline or DEX. Tail-cuff systolic blood pressure (SBP) was measured second daily. Kidney mitochondrial superoxide was examined using (MitoSOX) Red (MitoSOX) via flow cytometry. RESULTS: SBP was increased by ACTH (P < 0.0005) and DEX (P < 0.0005). Alpha-lipoic acid alone did not alter SBP. With alpha-lipoic acid pretreatment, SBP was increased by ACTH (P' < 0.005) but not by DEX. Alpha-lipoic partially prevented ACTH-HT (P' < 0.0005) and fully prevented DEX-HT (P' < 0.0005) but failed to reverse DEX-HT. ACTH and DEX did not increase MitoSOX signal. In ACTH-hypertensive rats, high-dose alpha-lipoic acid (100 mg/rat/day) did not decrease SBP further but raised MitoSOX signal (P < 0.001), suggesting prooxidant activity. CONCLUSION: Glucocorticoid-induced hypertension in rats is prevented by alpha-lipoic acid via mechanisms other than mitochondrial superoxide reduction.

Immune responsiveness during disease progression from acute rheumatic fever to chronic rheumatic heart disease.

Streptococcus pyogenes causes pharyngitis in school age children, which if left untreated causes acute rheumatic fever (ARF) that later progress toward rheumatic heart disease (RHD). The pathogenesis of this disease and its progression as post infectious squeal is not well understood. In this study, percentages of CD4(+) and CD8(+) T-cells were compared among patients of ARF, RHD and Chronic RHD using flow cytometer. The production of Th1/Th2 cytokines from serum and endothelial cells of damaged and normal heart valves was also analyzed using flow cytometer. Results showed an inverse proportion of CD4(+) and CD8(+) T-cell numbers in ARF and RHD patients. Cytokine assay demonstrated a switch-over from Th1 to Th2 type, as the disease progressed. We observed significantly high IL-6 in ARF patients and high TNF-α in early RHD patients which allowed us to construct a hypothesis, that, during initial infection phase, lot of antibodies are produced (via IL-6) and TNF-α has a role in disease progression and tissue damage during RHD phase.

Priming the immune system for heart disease: a perspective on group A streptococci.

Although immune responses against group A streptococci and the heart have been correlated with antibodies and T cell responses against cardiac myosin, there is no unifying hypothesis about carditis caused globally by many different serotypes. Our study identified disease-specific epitopes of human cardiac myosin in the development of rheumatic carditis in humans. We found that immune responses to cardiac myosin were similar in rheumatic carditis among a small sample of worldwide populations, in which immunoglobulin G targeted human cardiac myosin epitopes in the S2 subfragment hinge region within S2 peptides containing amino acid residues 842-992 and 1164-1272. An analysis of rheumatic carditis in a Pacific Islander family confirmed the presence of potential rheumatogenic epitopes in the S2 region of human cardiac myosin. Our report suggests that cardiac myosin epitopes in rheumatic carditis target the S2 region of cardiac myosin and are similar among populations with rheumatic carditis worldwide, regardless of the infecting group A streptococcal M serotype.

Differential induction of immunoregulatory circuits of phagocytic cells by Gal/Gal NAc lectin from pathogenic and nonpathogenic Entamoeba.

INTRODUCTION: Amoebic liver abscess (ALA) is an acute inflammatory disease caused by Entamoeba histolytica. MATERIALS AND METHODS: The key player in the pathogenesis and immunoprotection is the Gal/GalNAc inhibitable lectin, possessed by both pathogenic (Entamoeba histolytica) and nonpathogenic (Entamoeba dispar) strains, but only E. histolytica infection is associated with an acute inflammation and subsequently the disease. RESULTS AND DISCUSSION: The stimulation with the lectin induces the secretion of various proinflammatory cytokines/chemokines from intestinal epithelial cells. The differential induction of cytokine/chemokine network by the two strains can further regulate the immunoregulatory functions of the immune cells (monocytes and neutrophils) of the host. The soluble levels of IL-1beta, IL-6, IL-8, IL-10, MIP-1alpha, MCP-1, RANTES, GRO-alpha, GMCSF were quantitated to be significantly higher in pathogenic lectin-induced conditioned media (LCM) compared to nonpathogenic LCM (NP-LCM). The monocytes from ALA patients responded to the presence of pathogenic-LCM (P-LCM) by lowering of intracellular Ca(2+) (which was still higher than control). The proinflammatory MCP-1, GRO-alpha, and GMCSF levels in the monocytes were recorded both (quantitatively and mRNA quantitation) to be tremendously higher along with their respective receptors, with P-LCM compared to NP-LCM. A significant increase in the reactive oxygen intermediates and chemotactic index (CI) was observed in the monocytes when treated with P-LCM. Similarly, in neutrophils of ALA patients, an increase in intracellular Ca(2+), ROS and chemotaxis was observed with P-LCM. OBJECTIVES: The study is a step towards understanding the mechanism of immunopathogenesis of amoebiasis, on one hand, and points to the central role of cytokine/chemokine network in the process, on the other hand.