Identification of host immune-related biomarkers in active tuberculosis: A comprehensive analysis of differentially expressed genes.

Tuberculosis (TB) is a serious public health issue in India. Numerous molecular mechanisms and immunological responses play significant roles in the pathogenesis of tuberculosis. This study aimed to identify host immune-related biomarkers that are significantly differentially expressed in active TB and that play a vital role in disease progression. The methodology employed in this study included data collection, pre-processing, analysis, and interpretation of the results. Six microarray datasets were used to identify differentially expressed genes (DEGs), and only the common DEGs were used for further downstream analysis, such as hub gene identification, gene ontology, pathway enrichment analysis, and drug-gene interaction analysis. The study identified 1728 DEGs, including 906 upregulated and 822 downregulated genes. Five hub genes were identified that were: STAT1, GBP5, GBP1, FCGR1A, and BATF2. Gene ontology and pathway enrichment revealed that most of the genes were involved in interferon-gamma signaling. In addition, through drug-gene interactions, known drugs have been identified for STAT1, FCGR1A and GBP1. The findings of this study may contribute to early detection and treatment of active TB.

The Impact of Calcium Depletion on Proliferation of Chlorella sorokiniana Strain DSCG150.

This study analyzed the effects of Ca2+ metal ions among culture medium components on the Chlorella sorokiniana strain DSCG150 strain cell growth. The C. sorokiniana strain DSCG150 strain grew based on a multiple fission cell cycle and growth became stagnant in the absence of metal ions in the medium, particularly Ca2+. Flow cytometry and confocal microscopic image analysis results showed that in the absence of Ca2+, cell growth became stagnant as the cells accumulated into four autospores and could not transform into daughter cells. Genetic analysis showed that the absence of Ca2+ caused upregulation of calmodulin (calA) and cell division control protein 2 (CDC2_1) genes, and downregulation of origin of replication complex subunit 6 (ORC6) and dual specificity protein phosphatase CDC14A (CDC14A) genes. Analysis of gene expression patterns by qRT-PCR showed that the absence of Ca2+ did not affect cell cycle progression up to 4n autospore, but it inhibited Chlorella cell fission (liberation of autospores). The addition of Ca2+ to cells cultivated in the absence of Ca2+ resulted in an increase in n cell population, leading to the resumption of C. sorokiniana growth. These findings suggest that Ca2+ plays a crucial role in the fission process in Chlorella.

Alleviation of Salt Stress and Changes in Glycyrrhizic Acid Accumulation by Dark Septate Endophytes in Glycyrrhiza glabra Grown under Salt Stress.

This study aimed to investigate the mechanisms by which dark septate endophytes (DSE) regulate salt tolerance and the accumulation of bioactive constituents in licorice. First, the salt stress tolerance and resynthesis with the plant effect of isolated DSE from wild licorice were tested. Second, the performance of licorice inoculated with DSE, which had the best salt-tolerant and growth-promoting effects, was examined under salt stress. All isolated DSE showed salt tolerance and promoted plant growth, withCurvularia lunata D43 being the most effective. Under salt stress, C. lunata D43 could promote growth, increase antioxidant enzyme activities, enhance glycyrrhizic acid accumulation, improve key enzyme activities in the glycyrrhizic acid synthesis pathway, and induce the expression of the key enzyme gene and salt tolerance gene of licorice. The structural equation model demonstrated that DSE alleviate the negative effects of salt stress through direct and indirect pathways. Variations in key enzyme activities, gene expression, and bioactive constituent concentration can be attributed to the effects of DSE. These results contribute to revealing the value of DSE for cultivating medicinal plants in saline soils.

ATBS1-INTERACTING FACTOR 2 Positively Regulates Freezing Tolerance via INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR-Induced Cold Acclimation Pathway.

The INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR (ICE1/CBF) pathway plays a crucial role in plant responses to cold stress, impacting growth and development. Here, we demonstrated that ATBS1-INTERACTING FACTOR 2 (AIF2), a non-DNA-binding basic helix-loop-helix transcription factor, positively regulates freezing tolerance through the ICE1/CBF-induced cold tolerance pathway in Arabidopsis. Cold stress transcriptionally upregulated AIF2 expression and induced AIF2 phosphorylation, thereby stabilizing the AIF2 protein during early stages of cold acclimation. The AIF2 loss-of-function mutant, aif2-1, exhibited heightened sensitivity to freezing before and after cold acclimation. In contrast, ectopic expression of AIF2, but not the C-terminal-deleted AIF2 variant, restored freezing tolerance. AIF2 enhanced ICE1 stability during cold acclimation and promoted the transcriptional expression of CBFs and downstream cold-responsive genes, ultimately enhancing plant tolerance to freezing stress. MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK3/6), known negative regulators of freezing tolerance, interacted with and phosphorylated AIF2, subjecting it to protein degradation. Furthermore, transient co-expression of MPK3/6 with AIF2 and ICE1 downregulated AIF2/ICE1-induced transactivation of CBF2 expression. AIF2 interacted preferentially with BIN2 and MPK3/6 during the early and later stages of cold acclimation, respectively, thereby differentially regulating AIF2 activity in a cold acclimation time-dependent manner. Moreover, AIF2 acted additively in a gain-of-function mutant of BRASSINAZOLE-RESISTANT 1 (BZR1; bzr1-1D) and a triple knockout mutant of BRASSINOSTEROID-INSENSITIVE 2 (BIN2) and its homologs (bin2bil1bil2) to induce CBFs-mediated freezing tolerance. This suggests that cold-induced AIF2 coordinates freezing tolerance along with BZR1 and BIN2, key positive and negative components, respectively, of brassinosteroid signaling pathways.

Exploring the role of vitamin D-VDR pathway in pemphigus foliaceous: a novel perspective on disease pathogenesis.

Several auto-immune diseases have been linked to vitamin D deficiency as a contributing environmental factor. Its pleiotropic effects on the immune system, especially its essential role in maintaining immune tolerance, make the vitamin D pathway of great interest. In this study, we focused on Pemphigus foliaceous (PF) in Tunisian population. we aimed to quantify the Serum 25[OH]D levels using chemiluminescence assay and to analyze the differential expression of the VDR, CYP27B1 and CYP24A1 genes in the circulating blood cells and lesional skin tissue of PF patients using Q-PCR. A genetic explanation was then sought to explore any direct relationship between tag polymorphisms and the inherited features of PF. Results confirmed a vitamin D hypovitaminosis in Tunisian PF patients. Interestingly, a differential gene expression correlated to the disease stratification was noted. Indeed, at the systemic level, an upregulation of VDR and CYP27B1 genes was observed in healthy controls compared to PF patients. Notably, in lesional skin tissue, the clinical and serological remission phase was correlated with high transcriptional levels of the VDR gene and conversely a drop in expression of the CYP24A1 gene. Genetic analysis indicated the involvement of the most appealing polymorphisms, rs2228570 and poly (A) microsatellite, in PF etiopathogenesis. Indeed, CAC13 haplotype was associated with a higher risk of PF development. Our findings suggest that alterations in the vitamin D-VDR pathway may influence PF physiopathology, making this pathway a potential target for pharmacological modulation, especially for cortico-resistant PF patients.

Environmental stressor assessment of hydrocarbonoclastic bacteria biofilms from a marine oil spill.

The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, Micrococcus luteus and M. yunnanensis isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: M. luteus up to 98.79 % and M. yunnanensis 97.77 % removal. The assessment of Micrococcus biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in M. luteus by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in M. luteus than in M. yunnanensis. In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the uspA gene expression was analysed in Micrococcus biofilm under environmental stressors. The uspA expression increased up to 2.5-fold in M. luteus biofilms at 30 °C, and 1.3-fold at 50 °C. The highest uspA expression was recorded in M. yunnanensis biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. M. yunnanensis biofilms showed greater resilience than M. luteus biofilms when exposed to harsh environmental stressors. M. yunnanensis biofilms were thicker than M. luteus biofilms. Both biofilm responses to environmental stressors through uspA gene expression were consistent with the behaviours observed in the response surface analyses. The uspA gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.

Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots.

Synergy of gut microbiota and host genome in driving heterosis expression of chickens.

Heterosis has been widely utilized in agricultural production. Despite over a century of extensive research, the underlying mechanisms of heterosis remain elusive. Most hypotheses and research have focused on the genetic basis of heterosis. However, the potential role of gut microbiota in heterosis has been largely ignored. Here, we carefully design a crossbreeding experiment with two distinct broiler breeds and conduct 16S rRNA amplicon and transcriptome sequencing to investigate the synergistic role of gut microbiota and host genes in driving heterosis. We find that the breast muscle weight of the hybrids exhibits a high heterosis, 6.28% higher than mid-parent value. A notable difference is observed in the composition and potential function of cecal microbiota between hybrids and their parents. Over 90% of the differentially colonized microbiota and differentially expressed genes exhibit nonadditive patterns. Integrative analyses uncover associations between nonadditive genes and nonadditive microbiota, including a connection between the expression of cellular signaling pathway and metabolism-related genes and the abundance of Odoribacter, Oscillibacter, and Alistipes in hybrids. Moreover, higher abundances of these microbiota are related to better meat yield. In summary, these findings highlight the importance of gut microbiota in heterosis, serving as crucial factors that modulate heterosis expression in chickens.

Interplay between gut microbiome, host genetic and epigenetic modifications in MASLD and MASLD-related hepatocellular carcinoma.

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a wide spectrum of liver injuries, ranging from hepatic steatosis, metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis to MASLD-associated hepatocellular carcinoma (MASLD-HCC). Recent studies have highlighted the bidirectional impacts between host genetics/epigenetics and the gut microbial community. Host genetics influence the composition of gut microbiome, while the gut microbiota and their derived metabolites can induce host epigenetic modifications to affect the development of MASLD. The exploration of the intricate relationship between the gut microbiome and the genetic/epigenetic makeup of the host is anticipated to yield promising avenues for therapeutic interventions targeting MASLD and its associated conditions. In this review, we summarise the effects of gut microbiome, host genetics and epigenetic alterations in MASLD and MASLD-HCC. We further discuss research findings demonstrating the bidirectional impacts between gut microbiome and host genetics/epigenetics, emphasising the significance of this interconnection in MASLD prevention and treatment.

A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-ß protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.

Optimized Workflow from Gene to Product.

This chapter outlines the workflow using the ExpiSf™ Expression System designed for high-density infection of suspension ExpiSf9™ cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf™ chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf™ Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.

Generation of Complex Protein Structures by Coinfection with High-Quality Recombinant Baculovirus.

The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

Probing Baculovirus Vector Gene Essentiality for Foreign Gene Expression Using a CRISPR-Cas9 System.

The baculovirus expression vector system (BEVS) has now found acceptance in both research laboratories and industry, which can be attributed to many of its key features including the limited host range of the vectors, their non-pathogenicity to humans, and the mammalian-like post-translational modification (PTMs) that can be achieved in insect cells. In fact, this system acts as a middle ground between prokaryotes and higher eukaryotes to produce complex biologics. Still, industrial use of the BEVS lags compared to other platforms. We have postulated that one reason for this has been a lack of genetic tools that can complement the study of baculovirus vectors, while a second reason is the co-production of the baculovirus vector with the desired product. While some genetic enhancements have been made to improve the BEVS as a production platform, the genome remains under-scrutinized. This chapter outlines the methodology for a CRISPR-Cas9-based transfection-infection assay to probe the baculovirus genome for essential/nonessential genes that can potentially maximize foreign gene expression under a promoter of choice.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

Nonviral Platform for Expression of Recombinant Protein in Insect Cells.

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

SP110 Could be Used as a Potential Predictive and Therapeutic Biomarker for Oral Cancer.

The morbidity of oral squamous cell carcinoma (OSCC) has been rising year after year, making it a major global health issue. But the molecular pathogenesis of OSCC is currently unclear. To study the potential pathogenesis of OSCC, the differentially expressed genes (DEGs) were screened, and multiple databases were used to perform the tumor stage, expression, prognosis, protein-protein interaction (PPI) networks, modules, and the functional enrichment analysis. Moreover, we have identified SP110 as the key candidate gene and conducted various analyses on it using multiple databases. The research indicated that there were 211 common DEGs, and they were enriched in various GO terms and pathways. Meanwhile, one DEG is significantly related to short disease-free survival, four are associated with overall survival, and 12 DEGs have close ties with tumor staging. Additionally, the SP110 is significantly associated with methylation level, HPV status, tumor staging, gender, race, tumor grade, age, and overall/disease-free survival of oral cancer patients, as well as the immune process. The copy number variation of SP110 significantly affected the abundance of immune infiltration. Therefore, we speculate that SP110 could be used as the diagnostic and therapeutic biomarker for OSCC, and can help to further understand oral carcinogenesis.