Journal, genetic engineering & biotechnology最新文献

Background: The contribution of the processes involved and waste generated during gold mining to the increment of heavy metals concentration in the environment has been well established. While certain heavy metals are required for the normal functioning of an organism, certain heavy metals have been identified for their deleterious effects on the ecosystem and non-physiological roles in organisms. Hence, efforts aimed at reducing their concentration level are crucial. To this end, soil and water samples were collected from Ilesha gold mining, Osun State, Nigeria, and they were subjected to various analyses aimed at evaluating their various physicochemical parameters, heavy metal concentration, heavy metal-resistant bacteria isolation, and other analyses which culminated in the molecular characterization of heavy metal-resistant bacteria.

Results: Notably, the results obtained from this study revealed that the concentration of heavy metal in the water samples around the mining site was in the order Co > Zn > Cd > Pb > Hg while that of the soil samples was in the order Co > Cd > Pb > Hg > Zn. A minimum inhibitory concentration test performed on the bacteria isolates from the samples revealed some of the isolates could resist as high as 800 ppm of Co, Cd, and Zn, 400 ppm, and 100 ppm of Pb and Hg respectively. Molecular characterization of the isolates revealed them as Priestia aryabhattai and Enterobacter cloacae.

Conclusion: Further analysis revealed the presence of heavy metal-resistant genes (HMRGs) including merA, cnrA, and pocC in the isolated Enterobacter cloacae. Ultimately, the bacteria identified in this study are good candidates for bioremediation and merit further investigation in efforts to bioremediate heavy metals in gold mining sites.

Background: Staphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed.

Results: The genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature.

Conclusions: These findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.

Background: Human parainfluenza viruses (HPIVs) are common RNA viruses responsible for respiratory tract infections. Human parainfluenza virus 3 (HPIV-3) is particularly pathogenic, causing severe illnesses with no effective vaccine or therapy available.

Results: The current study employed a systematic immunoinformatic/reverse vaccinology approach to design a multiple epitope-based peptide vaccine against HPIV-3 by analyzing the virus proteome. On the basis of a number of therapeutic features, all three stable and antigenic proteins with greater immunological relevance, namely matrix protein, hemagglutinin neuraminidase, and RNA-directed RNA polymerase L, were chosen for predicting and screening suitable T-cell and B-cell epitopes. All of our desired epitopes exhibited no homology with human proteins, greater population coverage (99.26%), and high conservancy among reported HPIV-3 isolates worldwide. All of the T- and B-cell epitopes are then joined by putative ligands, yielding a 478-amino acid-long final construct. Upon computational refinement, validation, and thorough screening, several programs rated our peptide vaccine as biophysically stable, antigenic, allergenic, and non-toxic in humans. The vaccine protein demonstrated sufficiently stable interaction as well as binding affinity with innate immune receptors TLR3, TLR4, and TLR8. Furthermore, codon optimization and virtual cloning of the vaccine sequence in a pET32a ( +) vector showed that it can be readily expressed in the bacterial system.

Conclusion: The in silico designed HPIV-3 vaccine demonstrated potential in evoking an effective immune response. This study paves the way for further preclinical and clinical evaluation of the vaccine, offering hope for a future solution to combat HPIV-3 infections.

Background: Plant probiotics bacteria are live microbes that promote soil health and plant growth and build the stress-tolerant capacity to the plants. They benefit the plants by increasing nutrient absorption and release of stress-related phytohormones. These plant probiotic bacteria serve a better purpose to the plant when compared to chemical fertilizers. Use of chemical fertilizers such as arsenic and cadmium can lead to soil acidification and even release of harmful gases such as methane which further pollutes the environment.

Results: Different bacterial species were isolated from the agricultural fields of Tattiannaram, Telangana, and identified as the efficient rhizosphere bacteria with the essential qualities of plant growth promotion by evaluating the nitrogen-fixing ability on a selective media and various other methods. Upon the molecular characterization of the isolates, they were identified as Corynebacterium spp., Bacillus spp., Lactobacillus spp., and Cytobacillus spp. The results were also examined using various bioinformatics tools for accuracy in their phylogenetic pattern.

Conclusion: The recognized species of plant probiotics have established roles in promoting plant growth and strengthening plant immunity. This research introduces an innovative methodology for evaluating and investigating recently identified bacterial isolates, focusing on their distinctive plant probiotic attributes. Through harnessing the potential of advantageous microorganisms and comprehending their interaction with plants and soil, our objective is to formulate inventive approaches to elevate crop productivity, enhance soil richness, and foster environmentally sustainable and robust agricultural methodologies. These characteristics exhibit promising potential for future incorporation into plant systems, fortifying growth and development, and underscoring their distinctive significance within the realm of agriculture.

Background: Yellow fever is a mosquito-borne viral hemorrhagic disease transmitted by several species of virus-infected mosquitoes endemic to tropical regions of Central and South America and Africa. Earlier in the twentieth century, mass vaccination integrated with mosquito control was implemented to eradicate the yellow fever virus. However, regular outbreaks occur in these regions which pose a threat to travelers and residents of Africa and South America. There is no specific antiviral therapy, but there can be an effective peptide-based vaccine candidate to combat infection caused by the virus. Therefore, the study aims to design a multi-epitope-based subunit vaccine (MESV) construct against the yellow fever virus to reduce the time and cost using reverse vaccinology (RV) approach.

Methods: Yellow fever virus contains 10,233 nucleotides that encode for 10 proteins (C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) including 3 structural and 7 non-structural proteins. Structural proteins-precursor membrane protein (prM) and envelope protein (E)-were taken as a target for B cell and T cell epitope screening. Further, various immunoinformatics approaches were employed to FASTA sequences of structural proteins to retrieve B cell and T cell epitopes. MESV was constructed from these epitopes based on allergenicity, antigenicity and immunogenicity, toxicity, conservancy, and population coverage followed by structure prediction. The efficacy of the MESV construct to bind with human TLR-3, TLR-4, and TLR-8 were evaluated using molecular docking and simulation studies. Finally, in-silico cloning of vaccine construct was performed withpBR322 Escherichia coli expression system using codon optimization.

Results: Predicted epitopes evaluated and selected for MESV construction were found stable, non-allergenic, highly antigenic, and global population coverage of 68.03% according to in-silico analysis. However, this can be further tested in in-vitro and in-vivo investigations. Epitopes were sequentially merged to construct a MESV consisting of 393 amino acids using adjuvant and linkers. Molecular docking and simulation studies revealed stable and high-affinity interactions. Furthermore, in-silico immune response graphs showed effective immune response generation. Finally, higher CAI value ensured high gene expression of vaccine in the host cell.

Conclusion: The designed MESV construct in the present in-silico study can be effective in generating an immune response against the yellow fever virus. Therefore, to prevent yellow fever, it can be an effective vaccine candidate. However, further downstream, in-vitro study is required.

Background: One thousand sixty-one individuals were sampled from the cities of Anbar, Baghdad, Basra, Diyala, Najaf, and Wasit in Iraq and typed for 15 forensic STRs to explore the genetic structure of Iraq and develop a forensic DNA database. The total number of alleles that were identified was 203.

Result: Analyses of molecular variance (AMOVA) were then conducted Baghdad provides a good representation of the rest of the country, while Anbar is the most genetically distinct. The average heterozygosities of these loci was 0.779, homozygosities was 0.221, polymorphism information content was 0.77, power of discrimination was 0.927, and power of exclusion was 0.563. At these loci, a matching genotype will occur, on average, in 1 in 8.152 × 1017 individuals. For paternity tests, the average paternity probability for a matching profile is 99.9997%.

Conclusions: These loci are appropriate for use in forensic and paternity testing for this population. Iraq is similar to other countries in the Middle East, particularly Iran and Turkey, and is more similar to Europe than either Asia or Africa.

This article provides an overview of microbial host selection, synthetic biology, genome annotation, metabolic modeling, and computational methods for predicting gene essentiality for developing a microbial chassis. This article focuses on lactic acid bacteria (LAB) as a microbial chassis and strategies for genome annotation of the LAB genome. As a case study, Lactococcus lactis is chosen based on its well-established therapeutic applications such as probiotics and oral vaccine development. In this article, we have delineated the strategies for genome annotations of lactic acid bacteria. These strategies also provide insights into streamlining genome reduction without compromising the functionality of the chassis and the potential for minimal genome chassis development. These insights underscore the potential for the development of efficient and sustainable synthetic biology systems using streamlined microbial chassis with minimal genomes.

Background: The bark of Casuarina equisetifolia contains several active phytoconstituents that are suitable for the biosynthesis of gold nanoparticles (Au-NPs). These nanoparticles were subsequently evaluated for their effectiveness in reducing the toxicity induced by Chlorpyrifos (CPF) in rats.

Results: Various hematological and biochemical measurements were conducted in this study. In addition, markers of oxidative stress and inflammatory reactions quantified in liver and brain tissues were evaluated. Histopathological examinations were performed on both liver and brain tissues. Furthermore, the native electrophoretic protein and isoenzyme patterns were analyzed, and the relative expression levels of apoptotic genes in these tissues were determined. The hematological and biochemical parameters were found to be severely altered in the group injected with CPF. However, the administration of Au-C. equisetifolia nano-extract normalized these levels in all treated groups. The antioxidant system markers showed a significant decrease (P ≤ 0.05) in conjunction with elevated levels of inflammatory and fibrotic markers in both liver and brain tissues of the CPF-injected group. In comparison, the pre-treated group exhibited a reduction in these markers when treated with the nano-extract, as opposed to the CPF-injected group. Additionally, the nano-extract mitigated the severity of histopathological lesions induced by CPF in both liver and brain tissues, with a higher ameliorative effect observed in the pre-treated group. Electrophoretic assays conducted on liver and brain tissues revealed that the nano-extract prevented the qualitative changes induced by CPF in the pre-treated group. Furthermore, the molecular assay demonstrated a significant increase in the relative expression of apoptotic genes in the CPF-injected rats. Although the nano-extract ameliorated the relative expression of these genes compared to the CPF-injected group, it was unable to restore their values to normal levels.

Conclusion: Our results demonstrated that the nano-extract effectively reduced the toxicity induced by CPF in rats at hematological, biochemical, histopathological, physiological, and molecular levels, in the group pre-treated with the nano-extract.

Background: Lipases have emerged as essential biocatalysts, having the ability to contribute to a wide range of industrial applications. Microbial lipases have garnered significant industrial attention due to their stability, selectivity, and broad substrate specificity. In the previous study, a unique lipolytic bacterium (Micrococcus luteus EMP48-D) was isolated from tempeh. It turns out the bacteria produce an acidic lipase, which is important in biodiesel production. Our main objectives were to clone the acidic lipase and investigate its potential in biodiesel production.

Result: In this study, the gene encoding a lipase from M. luteus EMP48-D was cloned and expressed heterologously in Escherichia coli. To our knowledge, this is the first attempt at the cloning and expression of the lipase gene from Micrococcus luteus. The amino acid sequence was deduced from the nucleotide sequence (1356 bp) corresponded to a protein of 451 amino acid residues with a molecular weight of about 40 kDa. The presence of a signal peptide suggested that the protein was extracellular. A sequence analysis revealed that the protein had a lipase-specific Gly-X-Ser-X-Gly motif. The enzyme was identified as an acidic lipase with a pH preference of 5.0. Fatty acid preferences for enzyme activities were C8 and C12 (p-nitrophenyl esters), with optimum temperatures at 30-40 °C and still remaining active at 80°C. The enzyme was also shown to convert up to 70% of the substrate into fatty acid methyl ester.

Conclusion: The enzyme was a novel acidic lipase that demonstrated both hydrolytic and transesterification reactions. It appeared particularly promising for the synthesis of biodiesel as this enzyme's catalytic reaction was optimum at low temperatures and was still active at high temperatures.

Background: World Health Organization recommend the use of malaria vaccine, Mosquirix, as a malaria prevention strategy. However, Mosquirix has failed to reduce the global burden of malaria because of its inefficacy. The Mosquirix vaccine's modest effectiveness against malaria, 36% among kids aged 5 to 17 months who need at least four doses, fails to aid malaria eradication. Therefore, highly effective and efficacious malaria vaccines are required. The well-characterized P. falciparum circumsporozoite surface protein can be used to discover adjuvants that can increase the efficacy of Mosquirix. Therefore, the study sought to undertake an in-silico discovery of Plasmodium falciparum circumsporozoite surface protein inhibitors with pharmacological properties on Mosquirix using hierarchical virtual screening and molecular dynamics simulation.

Results: Monoclonal antibody L9, an anti-Plasmodium falciparum circumsporozoite surface protein molecule, was used to identify Plasmodium falciparum circumsporozoite surface protein inhibitors with pharmacological properties on Mosquirix during a virtual screening process in ZINCPHARMER that yielded 23 hits. After drug-likeness and absorption, distribution, metabolism, excretion, and toxicity property analysis in the SwissADME web server, only 9 of the 23 hits satisfied the requirements. The 9 compounds were docked with Plasmodium falciparum circumsporozoite surface protein using the PyRx software to understand their interactions. ZINC25374360 (-8.1 kcal/mol), ZINC40144754 (-8.3 kcal/mol), and ZINC71996727 (-8.9 kcal/mol) bound strongly to Plasmodium falciparum circumsporozoite surface protein with binding affinities of less than -8.0 kcal/mol. The stability of these molecularly docked Plasmodium falciparum circumsporozoite surface protein-inhibitor complexes were assessed through molecular dynamics simulation using GROMACS 2022. ZINC25374360 and ZINC71996727 formed stable complexes with Plasmodium falciparum circumsporozoite surface protein. They were subjected to in vitro validation for their inhibitory potential. The IC₅₀ values ranging between 250 and 350 ng/ml suggest inhibition of parasite development.

Conclusion: Therefore, the two Plasmodium falciparum circumsporozoite surface protein inhibitors can be used as vaccine adjuvants to increase the efficacy of the existing Mosquirix vaccine. Nevertheless, additional in vivo tests, structural optimization studies, and homogenization analysis are essential to determine the anti-plasmodial action of these adjuvants in humans.