Journal, genetic engineering & biotechnology最新文献

Background: The worldwide increase in human population and environmental damage has put immense pressure on the overall global crop production making it inadequate to feed the entire population. Therefore, the need for sustainable and environment-friendly practices to enhance agricultural productivity is a pressing priority. Endophytic bacteria with plant growth-promoting ability and biocontrol activity can strongly enhance plant growth under changing environmental biotic and abiotic conditions. Herein, we isolated halotolerant endophytic bacteria from an aquatic plant, Alternanthera philoxeroides, from the polluted waters of Madiwala Lake in Bangalore and studied their plant growth promotion (PGP) and biocontrol ability for use as bioinoculant.

Results: The isolated bacterial endophytes were screened for salt tolerance ranging from 5 to 15% NaCl concentration. Klebsiella pneumoniae showed halotolerant up to 10% NaCl and Bacillus amyloliquefaciens and Bacillus subtilis showed up to 15%. All three strains demonstrated good PGP abilities such as aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, phosphate solubilization, ammonia production, and nitrogen fixation. In addition, K. pneumoniae also exhibited high indoleacetic acid (IAA) production (195.66 ± 2.51 µg/ml) and potassium solubilization (2.13 ± 0.07 ppm). B. amyloliquefaciens and B. subtilis showed good extracellular enzyme production against cellulase, lipase, protease, and amylase. Both the isolates showed a broad spectrum of antimicrobial activity against the tested organisms. The optimization of IAA production by K. pneumoniae was done by the response surface methodology (RSM) tool. Characterization of IAA produced by the isolate was done by gas chromatography-mass spectrometry (GCMS) analysis. The enhanced plant growth-promoting ability of K. pneumoniae was also demonstrated using various growth parameters in a pot trial experiment using the seeds of Vigna unguiculata.

Conclusion: The isolated bacterial endophytes reported in this study can be utilized as PGP promotion and biocontrol agents in agricultural applications, to enhance crop yield under salinity stress. The isolate K. pneumoniae may be used as a biofertilizer in sustainable agriculture and more work can be done to optimize the best formulations for its application as a microbial inoculant for crops.

Vaccination is a crucial tool in preventing influenza, but it requires annual updates in vaccine composition due to the ever-changing nature of the flu virus. While healthcare and economic burdens have reduced, the virus remains a challenge. Research conducted over the past decade has revealed pathways for improvement through both basic and clinical studies. Viral surveillance plays a vital role in the better selection of candidate viruses for vaccines and the early detection of drug-resistant strains.This page offers a description of future vaccine developments and an overview of current vaccine options. In the coming years, we anticipate significant changes in vaccine production, moving away from traditional egg-based methods towards innovative technologies such as DNA and RNA vaccines. These newer approaches offer significant advantages over traditional egg-based and cell culture-based influenza vaccine manufacturing.

Background: Factorial design is a simple, yet elegant method to investigate the effect of multiple factors and their interaction on a specific response simultaneously. Hence, this type of study design reaches the best optimization conditions of a process. Although the interaction between the variables is widely prevalent in cell culture procedures, factorial design per se is infrequently utilized in improving cell culture output. Therefore, we aim to optimize the experimental conditions for generating mature bone marrow-derived dendritic cells (BMDCs). Two different variables were investigated, including the concentrations of the inducing factors and the starting density of the bone marrow mononuclear cells. In the current study, we utilized the design of experiments (DoE), a statistical approach, to systematically assess the impact of factors with varying levels on cell culture outcomes. Herein, we apply a two-factor, two-level (2²) factorial experiment resulting in four conditions that are run in triplicate. The two variables investigated here are cytokines combinations with two levels, granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or with interleukin-4 (IL4). The other parameter is cell density with two different concentrations, 2 × 10⁶ and 4 × 10⁶ cells/mL. Then, we measured cell viability using the trypan blue exclusion method, and a flow cytometer was used to detect the BMDCs expressing the markers FITC-CD80, CD86, CD83, and CD14. BMDC marker expression levels were calculated using arbitrary units (AU) of the mean fluorescence intensity (MFI).

Results: The current study showed that the highest total viable cells and cells yield obtained were in cell group seeded at 2 × 10⁶ cells/mL and treated with GM-CSF and IL-4. Importantly, the expression of the co-stimulatory molecules CD83 and CD80/CD86 were statistically significant for cell density of 2 × 10⁶ cells/mL (P < 0.01, two-way ANOVA). Bone marrow mononuclear cells seeded at 4 × 10⁶ in the presence of the cytokine mix less efficiently differentiated and matured into BMDCs. Statistical analysis via two-way ANOVA revealed an interaction between cell density and cytokine combinations.

Conclusion: The analysis of this study indicates a substantial interaction between cytokines combinations and cell densities on BMDC maturation. However, higher cell density is not associated with optimizing DC maturation. Notably, applying DoE in bioprocess designs increases experimental efficacy and reliability while minimizing experiments, time, and process costs.

Background: Cellulase is an important bioprocessing enzyme used in various industries. This study was conducted with the aim of improving the biodegradation activity of cellulase obtained from the Bacillus subtilis AG-PQ strain. For this purpose, AgO and FeO NPs were fabricated using AgNO₃ and FeSO₄·7H₂O salt respectively through a hydro-thermal method based on five major steps; selection of research-grade materials, optimization of temperature, pH, centrifuge, sample washed with distilled water, dry completely in the oven at the optimized temperature and finally ground for characterization. The synthesized NPs were characterized by scanning electron microscope (SEM), energy dispersive X-ray (EDX), and X-ray diffraction (XRD) to confirm the morphology, elemental composition, and structure of the sample respectively. The diameter of the NPs was recorded through SEM which lay in the range of 70-95 nm.

Results: Cultural parameters were optimized to achieve better cellulase production, where incubation time of 56 h, inoculum size of 5%, 1% coconut cake, 0.43% ammonium nitrate, pH 8, and 37 °C temperature were found optimal. The enhancing effect of AgO NPs was observed on cellulase activity (57.804 U/ml/min) at 50 ppm concentration while FeO NPs exhibited an inhibitory effect on cellulase activity at all concentrations. Molecular docking analysis was also performed to understand the underlying mechanism of improved enzymatic activity by nanocatalysts.

Conclusion: This study authenticates AgO NPs as better nanocatalysts for improved thermostable cellulase biodegradation activity with the extraordinary capability to be potentially utilized in bioethanol production.

Background: Brucellosis caused by B. melitensis is one of the most important common diseases between humans and livestock. Currently, live attenuated vaccines are used for this disease, which causes many problems, and unfortunately, there is no effective vaccine for human brucellosis. The aim of our research was to design a recombinant vaccine containing potential immunogenic epitopes against B. melitensis.

Methods: In this study, using immunoinformatics approaches, 3 antigens Omp31, Omp25, and Omp28 were identified and the amino acid sequence of the selected antigens was determined in NCBI. Signal peptides were predicted by SignaIP-5.0 server. To predict B-cell epitopes from ABCpred and Bcepred servers, to predict MHC-I epitopes from RANKPEP and SYFPEITHI servers, to predict MHC-II epitopes from RANKPEP and MHCPred servers, and to predict CTL epitopes were used from the CTLPred server. Potentially immunogenic final epitopes were joined by flexible linkers. Finally, allergenicity (AllerTOP 2.0 server), antigenicity (Vaxijen server), physicochemical properties (ProtParam server), solubility (Protein-sol server), secondary (PSIPRED and GRO4 servers) and tertiary structure (I-TASSER server), refinement (GalaxyWEB server), validation (ProSA-web, Molprobity, and ERRAT servers), and optimization of the codon sequence (JCat server) of the structure of the multi-epitope vaccine were analyzed.

Results: The analysis of immunoinformatics tools showed that the designed vaccine has high quality, acceptable physicochemical properties, and can induce humoral and cellular immune responses against B. melitensis bacteria. In addition, the high expression level of recombinant antigens in the E. coli host was observed through in silico simulation.

Conclusion: According to the results in silico, the designed vaccine can be a suitable candidate to fight brucellosis and in vitro and in vivo studies are needed to evaluate the research of this study.

Background: Aplastic anemia (AA) is a bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia which can lead to life-threatening complications. Our objective was to study the telomerase genes (TERT and TERC) variants, explore their relationship to telomere shortening and TERT gene expression, and to identify variants in the MPL gene within Egyptian AA patients.

Methods: Forty AA patients and 40 sex- and age-matched healthy individuals as the control group were studied through sequencing of TERT, TERC, and MPL genes. Quantitative real-time PCR (qRT-PCR) was used for measuring TERT gene expression. Telomere length (TL) was measured using the Quantitative Fluorescence In Situ Hybridization (Q-FISH) technique. In silico analysis was performed for the prediction of the pathogenicity of resultant variants.

Results: Sequencing of MPL, TERT, and TERC genes identified 26 variants. Eleven variants were identified in the MPL gene. Three of them are pathogenic: two missense [c.305 G>A, c.1589 C>T] and one splice site [g.9130T>G]. TERT gene sequencing showed thirteen variants, among them, four novel [c.484G>A, c.499G>A, c.512G>A, c.3164C>G] and two previously reported [c.835G>A, c.2031C>T] were predicted to be pathogenic. Two variants were characterized within the TERC gene; n.514A>G and n.463 C>T. TERT gene expression was downregulated in 70% of studied patients and the Q-FISH technique detected telomere shortening in 82.5% of patients.

Conclusions: Twenty-six pathogenic and benign variants within the TERC, TERT, and MPL genes were identified among the studied AA patients that were in several cases associated with shortened telomeres and/or lower TERT gene expression. Genotype/phenotype correlation in AA patients is of great importance in explaining the disease severity and guiding therapeutic decisions.

Background: The ongoing concern surrounding coronavirus disease 2019 (COVID-19) primarily stems from continuous mutations in the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), leading to the emergence of numerous variants. The receptor-binding domain (RBD) in the S1 subunit of the S protein of the virus plays a crucial role in recognizing the host's angiotensin-converting enzyme 2 (hACE2) receptor and facilitating cell membrane fusion processes, making it a potential target for preventing viral entrance into cells. This research aimed to determine the potential of banana lectin (BanLec) proteins to inhibit SARS-CoV-2 attachment to host cells by interacting with RBD through computational modeling.

Materials and methods: The BanLecs were selected through a sequence analysis process. Subsequently, the genes encoding BanLec proteins were retrieved from the Banana Genome Hub database. The FGENESH online tool was then employed to predict protein sequences, while web-based tools were utilized to assess the physicochemical properties, allergenicity, and toxicity of BanLecs. The RBDs of SARS-CoV-2 were modeled using the SWISS-MODEL in the following step. Molecular docking procedures were conducted with the aid of ClusPro 2.0 and HDOCK web servers. The three-dimensional structures of the docked complexes were visualized using PyMOL. Finally, molecular dynamics simulations were performed to investigate and validate the interactions of the complexes exhibiting the highest interactions, facilitating the simulation of their dynamic properties.

Results: The BanLec proteins were successfully modeled based on the RNA sequences from two species of banana (Musa sp.). Moreover, an amino acid modification in the BanLec protein was made to reduce its mitogenicity. Theoretical allergenicity and toxicity predictions were conducted on the BanLecs, which suggested they were likely non-allergenic and contained no discernible toxic domains. Molecular docking analysis demonstrated that both altered and wild-type BanLecs exhibited strong affinity with the RBD of different SARS-CoV-2 variants. Further analysis of the molecular docking results showed that the BanLec proteins interacted with the active site of RBD, particularly the key amino acids residues responsible for RBD's binding to hACE2. Molecular dynamics simulation indicated a stable interaction between the Omicron RBD and BanLec, maintaining a root-mean-square deviation (RMSD) of approximately 0.2 nm for a duration of up to 100 ns. The individual proteins also had stable structural conformations, and the complex demonstrated a favorable binding-free energy (BFE) value.

Conclusions: These results confirm that the BanLec protein is a promising candidate for developing a potential therapeutic agent for combating COVID-19. Furthermore, the results suggest the possibility of BanLec as a broad-spectrum antiv

Background: The health index of any population is directly correlated with the water quality, which in turn depends upon physicochemical characteristics and the microbiome of that aquatic source. For maintaining the water quality, knowledge of microbial diversity is a must. The present investigation attempts to evaluate the microflora of Baner. Metagenomics has been proven to be the technique for examining the genetic diversity of unculturable microbiota without using traditional culturing techniques. The microbial profile of Baner is analyzed using metagenomics for the first time to the best of our knowledge.

Results: To explore the microbial diversity of Baner, metagenomics analysis from 3 different sites was done. Data analysis identified 29 phyla, 62 classes, 131 orders, 268 families, and 741 genera. Proteobacteria was found to be the most abundant phylum in all the sampling sites, with the highest abundance at S₃ sampling site (94%). Bacteroidetes phylum was found to be second abundant in S₁ and S₂ site, whereas Actinobacteria was second dominant in sampling site S₃. Enterobacteriaceae family was dominant in site S1, whereas Comamonadaceae and Pseudomonadaceae was abundant in sites S₂ and S₃ respectively. The Baner possesses an abundant bacterial profile that holds great promise for developing bioremediation tactics against a variety of harmful substances.

Conclusion: Baner river's metagenomic analysis offers the first insight into the microbial profile of this hilly stream. Proteobacteria was found to be the most abundant phylum in all the sampling sites indicating anthropogenic interference and sewage contamination. The highest abundance of proteobacteria at S₃ reveals it to be the most polluted site, as it is the last sampling site downstream of the area under investigation, and falls after crossing the main city, so more human intervention and pollution were observed. Despite some pathogens, a rich profile of bacteria involved in bioremediation, xenobiotic degradation, and beneficial fish probiotics was observed, reflecting their potential applications for improving water quality and establishing a healthy aquaculture and fishery section.

Context: Marburg virus (MARV) is a member of the Filoviridae family and causes Marburg virus disease (MVD) among humans and primates. With fatality rates going up to 88%, there is currently no commercialized cure or vaccine to combat the infection. The National Institute of Allergy and Infectious Diseases (NIAID) classified MARV as priority pathogen A, which presages the need for a vaccine candidate which can provide stable, long-term adaptive immunity. The surface glycoprotein (GP) and fusion protein (FP) mediate the adherence, fusion, and entry of the virus into the host cell via the TIM-I receptor. Being important antigenic determinants, studies reveal that GP and FP are prone to evolutionary mutations, underscoring the requirement of a vaccine construct capable of eliciting a robust and sustained immune response. In this computational study, a reverse vaccinology approach was employed to design a combinatorial vaccine from conserved and antigenic epitopes of essential viral proteins of MARV, namely GP, VP24, VP30, VP35, and VP40 along with an endogenous protein large polymerase (L).

Methods: Epitopes for T-cell and B-cell were predicted using TepiTool and ElliPro, respectively. The surface-exposed TLRs like TLR2, TLR4, and TLR5 were used to screen high-binding affinity epitopes using the protein-peptide docking platform MdockPeP. The best binding epitopes were selected and assembled with linkers to design a recombinant multi-epitope vaccine construct which was then modeled in Robetta. The in silico biophysical and biochemical analyses of the recombinant vaccine were performed. The docking and MD simulation of the vaccine using WebGro and CABS-Flex against TLRs support the stable binding of vaccine candidates. A virtual immune simulation to check the immediate and long-term immunogenicity was carried out using the C-ImmSim server.

Results: The biochemical characteristics and docking studies with MD simulation establish the recombinant protein vaccine construct MarVax as a stable, antigenic, and potent vaccine molecule. Immune simulation studies reveal 1-year passive immunity which needs to be validated by in vivo studies.

Background: Members of Enterobacteriaceae such as Escherichia coli O 157:H7, Salmonella sp., Shigella sp., Klebsiella sp., and Citrobacter freundii are responsible for the outbreak of serious foodborne illness and other mucosal infections across the globe. The outer membrane proteins (OMPs) of Enterobacteriaceae are highly immunogenic in eliciting immune responses against pathogens. Moreover, the OMPs are highly conserved in the Enterobacteriaceae family. Sequence homology in the OMPs will ensure the presence of conserved immunodominant regions with predominant epitopes. The OmpL is such an immunogen that is highly conserved among the Enterobacteriaceae pathogens. In this study, we performed computational analysis on the outer membrane porin (Omp) L of prominent Enterobacteriaceae pathogens.

Results: Multiple sequence and structural alignment analysis have revealed that the OmpL protein is highly conserved among the selected Enterobacteriaceae pathogens. This amount of sequence and structural homology uncovered the conserved antibody binding B-cell epitopes in the OmpL protein. The B-cell epitopes predicted in the OmpL of Salmonella typhimurium are highly conserved among the other Enterobacteriaceae pathogens.

Conclusion: In conclusion, these conserved B-cell epitopes will vouch for the generation of heterologous humoral immune response in conferring cross protection against the Enterobacteriaceae pathogens and control their outbreaks across the globe.