Comparative Bioinformatic Analysis Reveals Conserved Regions in SARS-CoV-2 Genome for RAPID Pandemic Response.

In the face of the SARS-CoV-2 pandemic, characterized by the virus's rapid mutation rates, developing timely and targeted therapeutic and diagnostic interventions presents a significant challenge. This study utilizes bioinformatic analyses to pinpoint conserved genomic regions within SARS-CoV-2, offering a strategic advantage in the fight against this and future pathogens. Our approach has enabled the creation of a diagnostic assay that is not only rapid, reliable, and cost-effective but also possesses a remarkable capacity to detect a wide array of current and prospective variants with unmatched precision. The significance of our findings lies in the demonstration that focusing on these conserved genomic sequences can significantly enhance our preparedness for and response to emerging infectious diseases. By providing a blueprint for the development of versatile diagnostic tools and therapeutics, this research paves the way for a more effective global pandemic response strategy.

Beneficial Effect of Vitamin D on Non-Alcoholic Fatty Liver Disease (NAFLD) Progression in the Zebrafish Model.

A major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD) results from excessive liver fat accumulation. Vitamin D (VitD) plays multiple important roles in diverse physiologic processes. Here, we describe the role of VitD in the complex pathogenesis of NAFLD and explore the possible therapeutic role of VitD supplementation in NAFLD therapy. To compare the effect of VitD to other interventions such as low-calorie diet, we induced NAFLD in young adult zebrafish (Danio rerio, AB strain) and monitored the effects of VitD supplementation on the disease course. The zebrafish administered with high-dose VitD (1.25 µg) had significantly reduced liver fat compared to those that received low-dose VitD (0.049 µg) or caloric restriction. Gene expression analysis revealed that VitD downregulated several pathways that may play a role in NAFLD etiology, which affected fatty acid metabolism, vitamins and their cofactors, ethanol oxidation, and glycolysis. The pathway analysis revealed that the cholesterol biosynthesis pathway and the isoprenoid biosynthetic process pathway were significantly upregulated whereas the small molecule catabolic process pathway significantly downregulated following the exposure of NAFLD zebrafish model to high VitD dose. Therefore, our findings suggest the association of novel biochemical pathways with NAFLD and highlight the potential of VitD supplementation to reverse the severity of NAFLD, especially in younger people.

Genetics of psychic ability - A pilot case-control exome sequencing study.

INTRODUCTION: It is commonly believed that psychic ability, like many mental and physical traits, runs in families. This suggests the presence of a genetic component. If such a component were found, it would constitute a biological marker of psychic ability and inform environmental or pharmacologic means of enhancing or suppressing this ability. METHODS: A case-control study design was used to evaluate differences between psychic cases and non-psychic controls. Over 3,000 candidates globally were screened through two online surveys to locate people who claimed they and other family members were psychic. Measures of relevance to the claimed abilities (e.g., absorption, empathy, schizotypy) were collected and based on those responses, individuals with indications of psychotic or delusional tendencies were excluded from further consideration. Eligible candidates were then interviewed and completed additional screening tests. Thirteen individuals were selected as the final "psychic cases," and ten age-, sex-, and ethnicity-matched individuals with no claims of psychic ability were selected as controls. DNA from the saliva of these 23 participants was subjected to whole-exome sequencing. Two independent bioinformatics analyses were blindly applied to the sequenced data, one focusing exclusively on protein-coding sequences and another that also included some adjacent noncoding sequences. RESULTS: Sequencing data were obtained for all samples, except for one in the control group that did not pass the quality controls and was not included in further analyses. After unblinding the datasets, none of the protein-coding sequences (i.e., exons) showed any variation that discriminated between cases and controls. However, a difference was observed in the intron (i.e., non-protein-coding region) adjacent to an exon in the TNRC18 gene (Trinucleotide Repeat-Containing Gene 18 Protein) on chromosome 7. This variation, an alteration of GG to GA, was found in 7 of 9 controls and was absent from all psychic cases. DISCUSSION: The most conservative interpretation of these results is that they result from random population sampling. However, when the results are considered in relation to other lines of evidence, the results are more provocative. Further research is justified to replicate and extend these findings.

Detection of gene mutations and gene-gene fusions in circulating cell-free DNA of glioblastoma patients: an avenue for clinically relevant diagnostic analysis.

Glioblastoma (GBM) is the most common type of glioma and is uniformly fatal. Currently, tumour heterogeneity and mutation acquisition are major impedances for tailoring personalized therapy. We collected blood and tumour tissue samples from 25 GBM patients and 25 blood samples from healthy controls. Cell-free DNA (cfDNA) was extracted from the plasma of GBM patients and from healthy controls. Tumour DNA was extracted from fresh tumour samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We also collected 180 tumour DNA datasets from GBM patients publicly available at the TCGA/PANCANCER project. These data were analysed for mutations and gene-gene fusions that could be potential druggable targets. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL-1 ), as compared to healthy controls (1.4 ± 0.4 ng·mL-1 ) of the same average age. We identified unique mutations in the cfDNA and tumour DNA of each GBM patient, including some of the most frequently mutated genes in GBM according to the COSMIC database (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%). Using our gene-gene fusion database, ChiTaRS 5.0, we identified gene-gene fusions in cfDNA and tumour DNA, such as KDR-PDGFRA and NCDN-PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors such as imatinib, sunitinib, and sorafenib. Moreover, we identified BCR-ABL1 (in 8% of patients), COL1A1-PDGFB (8%), NIN-PDGFRB (8%), and FGFR1-BCR (4%) in cfDNA of patients, which can be targeted by analogues of imatinib. ROS1 fusions (CEP85L-ROS1 and GOPC-ROS1), identified in 8% of patient cfDNA, might be targeted by crizotinib, entrectinib, or larotrectinib. Thus, our study suggests that integrated analysis of cfDNA plasma concentration, gene mutations, and gene-gene fusions can serve as a diagnostic modality for distinguishing GBM patients who may benefit from targeted therapy. These results open new avenues for precision medicine in GBM, using noninvasive liquid biopsy diagnostics to assess personalized patient profiles. Moreover, repeated detection of druggable targets over the course of the disease may provide real-time information on the evolving molecular landscape of the tumour.

Modulation of Cellular CpG DNA Methylation by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gammaherpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types and in KSHV-infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hypermethylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation450K and MethylationEpic BeadChip arrays and identified panels of hyper- and hypomethylated cellular promoters in KSHV-infected cells. We combined our genome-wide methylation analysis with high-throughput RNA sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hypermethylation and upregulated genes with hypomethylation. In addition, we show that treating the cells with a demethylating agent leads to reexpression of these downregulated genes, indicating that, indeed, DNA methylation plays a role in the repression of these human genes. Comparison between de novo infection and PEL suggests that the virus induces initial hypermethylation followed by a slow increase in genome-wide hypomethylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.IMPORTANCE In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a few methylated promoters have been identified in KSHV-infected cells. Here, we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV-infected cells. We have identified many hyper- and hypomethylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive cells from uninfected cells and may serve as the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV-associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here, we show that treating PEL cells with a demethylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders.

Recent studies have determined that the microbiome has direct effects on behavior, and may be dysregulated in neurodevelopmental conditions. Considering that neurodevelopmental conditions, such as autism, have a strong genetic etiology, it is necessary to understand if genes associated with neurodevelopmental disorders, such as Shank3, can influence the gut microbiome, and if probiotics can be a therapeutic tool. In this study, we have identified dysregulation of several genera and species of bacteria in the gut and colon of both male and female Shank3 KO mice. L. reuteri, a species with decreased relative abundance in the Shank3 KO mice, positively correlated with the expression of gamma-Aminobutyric acid (GABA) receptor subunits in the brain. Treatment of Shank3 KO mice with L. reuteri induced an attenuation of unsocial behavior specifically in male Shank3 mice, and a decrease in repetitive behaviors in both male and female Shank3 KO mice. In addition, L. reuteri treatment affected GABA receptor gene expression and protein levels in multiple brain regions. This study identifies bacterial species that are sensitive to an autism-related mutation, and further suggests a therapeutic potential for probiotic treatment.

Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive basal-like breast cancers.

The discoidin domain receptor 1 (DDR1) is overexpressed in breast carcinoma cells. Low DDR1 expression is associated with worse relapse-free survival, reflecting its controversial role in cancer progression. We detected DDR1 on luminal cells but not on myoepithelial cells of DDR1+/+ mice. We found that DDR1 loss compromises cell adhesion, consistent with data that older DDR1-/- mammary glands had more basal/myoepithelial cells. Basal cells isolated from older mice exerted higher traction forces than the luminal cells, in agreement with increased mammary branches observed in older DDR1-/- mice and higher branching by their isolated organoids. When we crossed DDR1-/- mice with MMTV-PyMT mice, the PyMT/DDR1-/- mammary tumors grew faster and had increased epithelial tension and matricellular fibrosis with a more basal phenotype and increased lung metastases. DDR1 deletion induced basal differentiation of CD90+CD24+ cancer cells, and the increase in basal cells correlated with tumor cell mitoses. K14+ basal cells, including K8+K14+ cells, were increased adjacent to necrotic fields. These data suggest that the absence of DDR1 provides a growth and adhesion advantage that favors the expansion of basal cells, potentiates fibrosis, and enhances necrosis/hypoxia and basal differentiation of transformed cells to increase their aggression and metastatic potential.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

BACKGROUND: Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. RESULTS: Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. CONCLUSIONS: These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. REVIEWERS: Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

Tissue Transglutaminase contributes to experimental multiple sclerosis pathogenesis and clinical outcome by promoting macrophage migration.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.

Cyclosporin-A-induced prion protein aggresomes are dynamic quality-control cellular compartments.

Despite the activity of cellular quality-control mechanisms, subsets of mature and newly synthesized polypeptides fail to fold properly and form insoluble aggregates. In some cases, protein aggregation leads to the development of human neurodegenerative maladies, including Alzheimer's and prion diseases. Aggregates of misfolded prion protein (PrP), which appear in cells after exposure to the drug cyclosporin A (CsA), and disease-linked PrP mutants have been found to accumulate in juxtanuclear deposition sites termed 'aggresomes'. Recently, it was shown that cells can contain at least two types of deposition sites for misfolded proteins: a dynamic quality-control compartment, which was termed 'JUNQ', and a site for terminally aggregated proteins called 'IPOD'. Here, we show that CsA-induced PrP aggresomes are dynamic structures that form despite intact proteasome activity, recruit chaperones and dynamically exchange PrP molecules with the cytosol. These findings define the CsA-PrP aggresome as a JUNQ-like dynamic quality-control compartment that mediates the refolding or degradation of misfolded proteins. Together, our data suggest that the formation of PrP aggresomes protects cells from proteotoxic stress.

Conditional modulation of membrane protein expression in cultured cells mediated by prion protein recognition of short phosphorothioate oligodeoxynucleotides.

We demonstrate that the levels of native as well as transfected prion protein (PrP) are lowered in various cell lines exposed to phosphorothioate oligodeoxynucleotides (PS-DNA) and can be rapidly reverted to their normal amounts by removal of PS-DNA. This transient modulation was independent of the glycosylation state of PrP, and in addition, all three PrP glycoforms were susceptible to PS-DNA treatment. Deletion of the N-terminal domain (amino acids 23-99), but not of the other domains of PrP, abrogated its PS-DNA-mediated down-regulation. PrP versions localized in the mitochondria, cytoplasm, or nucleus were not modulated by PS-DNA, indicating that PrP surface exposure is required for executing this effect. Proteins that in their native forms were not responsive to PS-DNA, such as thymocyte antigen 1 (Thy1), Doppel protein (Dpl), green fluorescent protein (GFP), and cyan fluorescent protein (CFP), became susceptible to PS-DNA-mediated down-regulation following introduction of the N terminus of PrP into their sequence. These observations demonstrate the essential role of the N-terminal domain for promoting oligonucleotide-mediated reduction of the PrP level and suggest that transient treatment of cultured cells with PS-DNA may provide a general method for targeted modulation of the levels of desired surface proteins in a conditional and reversible manner.

Phosphorothioate oligonucleotides reduce PrP levels and prion infectivity in cultured cells.

Prions are composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process. Here we report that short phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels was independent of the nucleotide sequence. The effective concentration (EC50) of PS-DNA required to achieve half-maximal diminution of PrPSc was approximately 70 nM, whereas the EC50 of PS-DNA for PrPC was more than 50-fold greater. This finding indicated that diminished levels of PrPSc after exposure to PS-DNA are unlikely to be due to decreased PrPC levels. Bioassays in transgenic mice demonstrated a substantial diminution in the prion infectivity after ScN2a cells were exposed to PS-DNAs. Whether PS-DNA will be useful in the treatment of prion disease in people or livestock remains to be established.

Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine.

An expanded polyglutamine domain in huntingtin underlies the pathogenic events in Huntington disease (HD), characterized by chorea, dementia and severe weight loss, culminating in death. Transglutaminase (TGase) may be critical in the pathogenesis, via cross-linking huntingtin. Administration of the TGase competitive inhibitor, cystamine, to transgenic mice expressing exon 1 of huntingtin containing an expanded polyglutamine repeat, altered the course of their HD-like disease. Cystamine given intraperitoneally entered brain where it inhibited TGase activity. When treatment began after the appearance of abnormal movements, cystamine extended survival, reduced associated tremor and abnormal movements and ameliorated weight loss. Treatment did not influence the appearance or frequency of neuronal nuclear inclusions. Unexpectedly, cystamine treatment increased transcription of one of the two genes shown to be neuroprotective for polyglutamine toxicity in Drosophila, dnaj (also known as HDJ1 and Hsp40 in humans and mice, respectively). Inhibition of TGase provides a new treatment strategy for HD and other polyglutamine diseases.

Evidence for a role for transglutaminase in Huntington's disease and the potential therapeutic implications.

Transglutaminase (TGase) activity is increased in affected regions of brains from patients with Huntington's disease (HD). TGase activity is particularly elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl-lysyl cross-links have been detected in nuclear inclusions in HD brain, indicating that TGase may play a prominent role in the aggregation of huntingtin (htt). Attempts to ameliorate experimental disease, via inhibition of TGase in transgenic models of HD in mice, are under investigation.