Gene Networks Analysis of Salmonella Typhimurium Reveals New Insights on Key Genes Involved in Response to Low Water Activity.

Background: When Salmonella enterica serovar Typhimurium, a foodborne bacterium, is exposed to osmotic stress, cellular adaptations increase virulence severity and cellular survival. Objectives: The aim of the gene network analysis of S. Typhimurium was to provide insights into the various interactions between the genes involved in cellular survival under low water activity (aw). Materials and Methods: We performed a gene network analysis to identify the gene clusters and hub genes of S. Typhimurium using Cytoscape in three food samples subjected to aw stress after 72 hours. Results: The identified hub genes of S. Typhimurium belonged to down-regulated genes and were related to translation, transcription, and ribosome structure in the food samples. The rpsB and Tig were identified as the most important of the hub genes. Enrichment analysis of the hub genes also revealed the importance of translation and cellular protein metabolic processes. Moreover, the biological process associated with organonitrogen metabolism in milk chocolate was identified. According to the KEGG pathway results of gene cluster analysis, cellular responses to stress were associated with RNA polymerase, ribosome, and oxidative phosphorylation. Genes encoding RNA polymerase activity, including rpoA, rpoB, and rpoZ, were also significantly identified in the KEGG pathways. The identified motifs of hub DEGs included EXPREG_00000850, EXPREG_00000b00, EXPREG_000008e0, and EXPREG_00000850. Conclusion: Based on the results of the gene network analysis, the identified hub genes may contribute to adaptation to food compositions and be responsible for the development of low water stress tolerance in Salmonella. Among the food samples, the milk chocolate matrix leads to more adaptation pathways for S. Typhimurium survival, as more hub genes were down-regulated and more motifs were detected. The identified motifs were involved in carbohydrate metabolism, carbohydrate transport, electron transfer, and oxygen transfer.

Regulatory non-coding RNA: The core defense mechanism against plant pathogens.

Plant pathogens damage crops and threaten global food security. Plants have evolved complex defense networks against pathogens, using crosstalk among various signaling pathways. Key regulators conferring plant immunity through signaling pathways include protein-coding genes and non-coding RNAs (ncRNAs). The discovery of ncRNAs in plant transcriptomes was first considered "transcriptional noise". Recent reviews have highlighted the importance of non-coding RNAs. However, understanding interactions among different types of noncoding RNAs requires additional research. This review attempts to consider how long-ncRNAs, small-ncRNAs and circular RNAs interact in response to pathogenic diseases within different plant species. Developments within genomics and bioinformatics could lead to the further discovery of plant ncRNAs, knowledge of their biological roles, as well as an understanding of their importance in exploiting the recent molecular-based technologies for crop protection.

Highlight of potential impact of new viral genotypes of SARS-CoV-2 on vaccines and anti-viral therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of the coronavirus disease (COVID-19) pandemic, has infected millions of people globally. Genetic variation and selective pressures lead to the accumulation of single nucleotide polymorphism (SNP) within the viral genome that may affect virulence, transmission rate, viral recognition and the efficacy of prophylactic and interventional measures. To address these concerns at the genomic level, we assessed the phylogeny and SNPs of the SARS-CoV-2 mutant population collected to date in Iran in relation to globally reported variants. Phylogenetic analysis of mutant strains revealed the occurrence of the variants known as B.1.1.7 (Alpha), B.1.525 (Eta), and B.1.617 (Delta) that appear to have delineated independently in Iran. SNP analysis of the Iranian sequences revealed that the mutations were predominantly positioned within the S protein-coding region, with most SNPs localizing to the S1 subunit. Seventeen S1-localizing SNPs occurred in the RNA binding domain that interacts with ACE2 of the host cell. Importantly, many of these SNPs are predicted to influence the binding of antibodies and anti-viral therapeutics, indicating that the adaptive host response appears to be imposing a selective pressure that is driving the evolution of the virus in this closed population through enhancing virulence. The SNPs detected within these mutant cohorts are addressed with respect to current prophylactic measures and therapeutic interventions.

Comparative phylogenetic analysis of SARS-CoV-2 spike protein-possibility effect on virus spillover.

Coronavirus disease 2019 has developed into a dramatic pandemic with tremendous global impact. The receptor-binding motif (RBM) region of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to host angiotensin-converting enzyme 2 (ACE2) receptors for infection. As ACE2 receptors are highly conserved within vertebrate species, SARS-CoV-2 can infect significant animal species as well as human populations. An analysis of SARS-CoV-2 genotypes isolated from human and significant animal species was conducted to compare and identify mutation and adaptation patterns across different animal species. The phylogenetic data revealed seven distinct phylogenetic clades with no significant relationship between the clades and geographical locations. A high rate of variation within SARS-CoV-2 mink isolates implies that mink populations were infected before human populations. Positions of most single-nucleotide polymorphisms (SNPs) within the spike (S) protein of SARS-CoV-2 genotypes from the different hosts are mostly accumulated in the RBM region and highlight the pronounced accumulation of variants with mutations in the RBM region in comparison with other variants. These SNPs play a crucial role in viral transmission and pathogenicity and are keys in identifying other animal species as potential intermediate hosts of SARS-CoV-2. The possible roles in the emergence of new viral strains and the possible implications of these changes, in compromising vaccine effectiveness, deserve urgent considerations.

Quasi-species nature and differential gene expression of severe acute respiratory syndrome coronavirus 2 and phylogenetic analysis of a novel Iranian strain.

A novel coronavirus related to severe acute respiratory syndrome virus, (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. Despite the genetic mutations across the SARS-CoV-2 genome being recently investigated, its transcriptomic genetic polymorphisms at inter-host level and the viral gene expression level based on each Open Reading Frame (ORF) remains unclear. Using available High Throughput Sequencing (HTS) data and based on SARS-CoV-2 infected human transcriptomic data, this study presents a high-resolution map of SARS-CoV-2 single nucleotide polymorphism (SNP) hotspots in a viral population at inter-host level. Four throat swab samples from COVID-19 infected patients were pooled, with RNA-Seq read retrieved from SRA NCBI to detect 21 SNPs and a replacement across the SARS-CoV-2 genomic population. Twenty-two RNA modification sites on viral transcripts were identified that may cause inter-host genetic diversity of this virus. In addition, the canonical genomic RNAs of N ORF showed higher expression in transcriptomic data and reverse transcriptase quantitative PCR compared to other SARS-CoV-2 ORFs, indicating the importance of this ORF in virus replication or other major functions in virus cycle. Phylogenetic and ancestral sequence analyses based on the entire genome revealed that SARS-CoV-2 is possibly derived from a recombination event between SARS-CoV and Bat SARS-like CoV. Ancestor analysis of the isolates from different locations including Iran suggest shared Chinese ancestry. These results propose the importance of potential inter-host level genetic variations to the evolution of SARS-COV-2, and the formation of viral quasi-species. The RNA modifications discovered in this study may cause amino acid sequence changes in polyprotein, spike protein, product of ORF8 and nucleocapsid (N) protein, suggesting further insights to understanding the functional impacts of mutations in the life cycle and pathogenicity of SARS-CoV-2.

Developing an Ephestia kuehniella Hemocyte Cell Line to Assess the Bio-Insecticidal Potential of Microencapsulated Helicoverpa armigera Nucleopolyhedrovirus Against Cotton Bollworm (Lepidoptera: Noctuidae) Larva.

Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) (genus: Alphabaculovirus, incertae sedis: Baculoviridae) has been used to control Helicoverpa armigera (Hübner). A reproducible and susceptible cell line was prepared from the hemocytes of Ephestia kuehniella in Grace and Ex-Cell 420 media. The population doubling time of these cloned cell cultures during the logarithmic phase were about 2.3 and 3.7 d for Ex-Cell 420 and Grace's media, respectively. When 60% confluence occurred, cells were infected by viral inoculums. All biochemical compounds were significantly changed relevant to cellular metabolism due to HearNPV infection. In order to improve its stability, two polymer formulations were used, i.e., formulation A (sodium alginate, gelatin, starch, and molasses) and formulation B (cottonseed kernel extract, Bran, glycerol, boric acid, egg white, and sugar). Formulant A provided high photostability by exhibiting 83.2 ± 3% efficacy and 88.66 ± 2.1% original activities remaining after 72 h UV exposure. Percentage original activity remaining of unformulated HearNPV and formulated mixture of B was 38.66 ± 2.6% and 9.33 ± 1.3%, respectively, after 72 h UV-irradiation. The virulence of the HearNPV proliferated from the Ex-Cell medium was similar to the virulence of wild-type HearNPV with LC50 of 7.7×105 OBs/ml. Formulant A, revealed only 20.0 ± 1% reduction in efficacy while the unformulated virus and formulant B faced a reduction of 90.0 ± 3% and 64.0 ± 2% after 72 h of UVA irradiation. Formulant A thus showed a high potential to protect HearNPVs microparticles against UV-inactivation suggesting a new platform for more efficient biological-management of cotton bollworm (specific name Helicoverpa armigera, genus: Helicoverpa, Lepidoptera: Noctuidae) in vivo.

Comparison of Protein Gut Samples from Rhipicephalus spp. Using a Crude and an Innovative Preparation Method for Proteome Analysis.

Tick populations are controlled through the application of chemical pesticides. However, the rise in chemical resistance has prompted the investigation of other control methods such as the use of tick vaccines. Proteomic analysis provides valuable information about the possible function and localization of proteins, as candidate vaccine proteins are often either secreted or localized on the cell-surface membrane. Progress in the utilization of proteomics for the identification of novel treatment targets has been significant. However, their use in tick-specific investigations is still quite novel, with the continual development of tick-specific methodologies essential. In this study, an innovative sample preparation method was utilized to isolate epithelial cells from tick midguts to identify the membrane-bound proteins. Proteomic analysis was conducted comparing crude and innovative sample preparation methods with 692 and 1242 tick-specific proteins, 108 and 314 surface proteins respectively, isolated from the midguts of semi-engorged Rhipicephalus microplus adult female ticks. This research reports a novel preparation protocol for the analysis of tick midgut proteins which reduces host protein contamination.

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus.

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2-HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.

Extracellular expression of the HT1 neurotoxin from the Australian paralysis tick in two Saccharomyces cerevisiae strains.

Surface display libraries (SDL) have predominantly been utilized for the screening of peptides, and single-chain variable IgG fragments, however, the use of SDL for the expression and purification of proteins is gaining interest. Prokaryote SDL express proteins within the periplasm, limiting the application of common screening techniques, such as ELISA and FACS, to assess the viability of recombinant toxin before purification. A previous attempt to express a functional holocyclotoxin-1 (HT1) from the Australian paralysis tick (Ixodes holocyclus) using a prokaryotic system was unsuccessful. In this study, the coding sequence (CDS) of HT1 was cloned into the pYD1 plasmid and transformed by electroporation into IMTV014 and EBY100 yeast cell lines. Post induction, recombinant HT1 was identified on the cell surface of IMTV014/ht1 and EBY100/ht1 transformants by FACS, Western blot, and ELISA utilizing dog anti-paralysis tick IgG. The recombinant HT1 was purified, and functionality confirmed by an in vitro synaptosome-binding assay. This research reports for the first time the extracellular expression and display of a functional HT1 on the surface of the S. cerevisiae. It also provides evidence that yeast display libraries provide a viable technology to produce recombinant toxins, and their screening using high throughput methodologies such as FACS.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells.

Rhipicephalus microplus - the cattle tick - is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.