Journal, genetic engineering & biotechnology最新文献

Background: Viral infections cause damage and long-term injury to infected human tissues, demanding therapy with antiviral and wound healing medications. Consequently, safe phytochemical molecules that may control viral infections with an ability to provide wound healing to viral-induced tissue injuries, either topically or systemically, are advantageous. Herein, we hypothesized that epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, might be effective as a wound healing, antiviral, and antifibrotic therapy.

Results: The antiviral activities of EGCG against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Herpes simplex virus type 2 (HSV-2) as well as its wound healing activities against different monolayer tissue (continuous and primary) systems were investigated. Consider its possible wound-healing advantages as well. To determine the safe concentrations of EGCG in green monkey kidney (Vero) and Vero-E6 cell lines, MTT assay was performed and showed high CC₅₀ values of 405.1 and 322.9 μM, respectively. The antiviral activities of EGCG against SARS-CoV-2 and HSV-2, measured as half-maximal concentration 50 (IC₅₀) concentrations, were 36.28 and 59.88 μM, respectively. These results confirm that the EGCG has remarkable viral inhibitory activities and could successfully suppress the replication of SARS-CoV-2 and HSV-2 in vitro with acceptable selectivity indices (SI) of 11.16 and 5.39, respectively. In parallel, the EGCG exhibits significant and dose/time-dependent anti-migration effects in human breast cancer cells (MCF-7), its resistant variation (MCF-7^adr), and human skin fibroblast (HSF) indicating their potential to heal injuries in different internal and topical mammalian systems.

Conclusions: The EGCG has proven to be an efficient antiviral against SARS-CoV-2 and HSV-2, as well as a wound-healing phytochemical. We assume that EGCG may be a promising option for slowing the course of acute cellular damage induced by systemic (Coronavirus Disease 2019 (COVID-19)) or topical (HSV-2) viral infections.

Background: The ability of actinomycetes to produce bioactive secondary metabolites makes them one of the most important prokaryotes. Marine actinomycetes are one of the most important secondary metabolites producers used for pharmaceuticals and other different industries.

Results: In this study, the promising actinomycetes were isolated from Abu-Qir Bay. Four different media named as starch nitrate, starch casein, glycerol asparagine, and glycerol glycine were used as a preliminary experimental media to study the role of the medium components on the counts of actinomycetes in sediment samples. The results indicated that starch casein medium reported the highest counts (30-63 CFU/g) in all the tested sites. Lower counts were detected on starch nitrate and glycerol asparagine. On the other hand, glycerol glycine medium gave the lowest counts (15-48 CFU/g). Abu-Qir₈ harbored the highest average count of actinomycetes (63 CFU/g), followed by Abu-Qir₁ (48 CFU/g). The lower counts were detected in Abu-Qir₅ and Abu-Qir₇ (26 and 29 CFU/g, respectively). A total of 12 pure obtained actinomycetes isolates were subjected to morphological, physiological, and biochemical characterization. The selected actinobacterial isolates were subjected to numerical analysis, and the majority of isolates were grouped into four main clusters (A, B, C, & D), and each of them harbored two isolates; additionally, four isolates did not cluster at this similarity level. Isolate W4 was carefully chosen as the most promising pigment and antimicrobial agent's producer; the produced pigment was extracted and optimized by statistical experiments (PBD & BBD) and was tested for its anti-inflammatory activity. The results showed anti-inflammatory effect and prevented the denaturation of BSA protein at a concentration much higher than the safe dose and increased with increasing the pigment concentration.

Conclusion: Marine actinomycetes play a vital role in the production of novel and important economic metabolites that have many industrial and pharmaceuticals applications. Streptomyces genera are the most important actinomycetes that produce important metabolites as previously reported.

Background: Phytase supplementation in rations can reduce their phytic acid composition in order to enhance their nutritional value. Aspergillus niger is a fungus that can encode phytase. This study aims to determine the characteristics of its DNA sequences and amino acid composition that encode the phytase enzyme, as well as to determine the primer designs.

Method: This study used gene sequence data and protein-encoding phytase from Aspergillus niger that was collected manually from NCBI and PDB. The data was analyzed using SPDBV and then be aligned using the ClustalW Multiple Alignment features. The phylogenetic tree was built by Mega11 software. Primers were designed from selected candidate sequences that were analyzed. The designed primers were then simulated for PCR using FastPCR and SnapGene software.

Results: There are 18 Aspergillus niger phytases in NCBI which is 14.87% of the total Aspergillus. There are 14 Aspergillus niger phytases that have identity above 95%. Aspergillus niger 110. M94550.1 is the closest strain to the PDB template. Candidate sources of phytase genes are Aspergillus niger 110.M94550.1, 48.2.BCMY01000003.1, and 92.JQ654450.1. The primer design has 2 possibilities of self-annealing and high melting temperature on the reverse primer. PCR simulation shows that the primer design can attach completely but still has the possibility of mispriming.

Conclusion: This study suggests promising results for the future development of phytase enzyme production from Aspergillus niger as a feed additive using genetic engineering to enhance the quality of livestock feed in Indonesia.

Background: Microsatellites or simple sequence repeats (SSR) consist of 1-6 nucleotide motifs of DNA or RNA which are ubiquitously present in tandem repeated sequences across genome in viruses: prokaryotes and eukaryotes. They may be localized to both the coding and non-coding regions. SSRs play an important role in replication, gene regulation, transcription, and protein function. The Caliciviridae (CLV) family of viruses have ss-RNA, non-enveloped, icosahedral symmetry 27-35 nm in diameter in size. The size of the genome lies between 6.4 and 8.6 kb.

Results: The incidence, composition, diversity, complexity, and host range of different microsatellites in 62 representatives of the family of Caliciviridae were systematically analyzed. The full-length genome sequences were assessed from NCBI ( https://www.ncbi.nlm.nih.gov ), and microsatellites were extracted through MISA software. The average genome size is about 7538 bp ranging from 6273 (CLV61) to 8798 (CLV47) bp. The average GC content of the genomes was ~ 51%. There are a total of 1317 SSRs and 53 cSSRs in the studied genomes. CLV 41 and CLV 49 contain the highest and lowest value of SSRs with 32 and 10 respectively, while CLV16 had maximum cSSR incidence of 4. There were 29 species which do not contain any cSSR. The incidence of mono-, di-, and tri-nucleotide SSRs was 219, 884, and 206, respectively. The most prevalent mono-, di-, and tri-nucleotide repeat motifs were "C" (126 SSRs), AC/CA (240 SSRs), and TGA/ACT (23 SSRs), respectively. Most of the SSRs and cSSRs are biased toward the coding region with a minimum of ~ 90% incident SSRs in the genomes' coding region. Viruses with similar host are found close to each other on the phylogenetic tree suggesting virus host being one of the driving forces for their evolution.

Conclusions: The Caliciviridae genomes does not conform to any pattern of SSR signature in terms of incidence, composition, and localization. This unique property of SSR plays an important role in viral evolution. Clustering of similar host in the phylogenetic tree is the evidence of the uniqueness of SSR signature.

Background: Cholesterol oxidase has numerous biomedical and industrial applications. In the current study, a new bacterial strain was isolated from sewage and was selected for its high potency for cholesterol degradation (%) and production of high cholesterol oxidase activity (U/OD₆₀₀).

Results: Based on the sequence of 16S rRNA gene, the bacterium was identified as Bacillus subtilis. The fermentation conditions affecting cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD₆₀₀) of B. subtilis were optimized through fractional factorial design (FFD) and response surface methodology (RSM). According to this sequential optimization approach, 80.152% cholesterol degradation was achieved by setting the concentrations of cholesterol, inoculum size, and magnesium sulphate at 0.05 g/l, 6%, and 0.05 g/l, respectively. Moreover, 85.461 U of cholesterol oxidase/OD₆₀₀ were attained by adjusting the fermentation conditions at initial pH, 6; volume of the fermentation medium, 15 ml/flask; and concentration of cholesterol, 0.05 g/l. The optimization process improved cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD₆₀₀) by 139% and 154%, respectively. No cholesterol was detected in the spectroscopic analysis of the optimized fermented medium via gas chromatography-mass spectroscopy (GC-MS).

Conclusion: The current study provides principal information for the development of efficient production of cholesterol oxidase by B. subtilis that could be used in various applications.

Background: The presence of drug-resistant Gram-negative pathogenic bacteria and Extended Spectrum β-Lactamase Producers (ESBLs) in hospital associated fomites like door handles can serve as vehicles in transmission and may be the key factor in epidemiology of ESBL producing bacterial infection not only in a hospital setting but also in the community. The aim of this study was to determine the prevalence of ESBLs and antibiotic resistance of Gram-Negative pathogenic Bacteria isolated from door-handles in two selected hospitals in Pokhara Metropolitan City, Nepal. The study was conducted in selected hospitals in Pokhara Metropolitan City, Western Nepal. A cross-sectional study design was used. The hospitals were selected randomly. A total of 100 swab samples were taken from door-handles. Isolation and identification of bacteria were done using standard microbiological procedures. An antibiotic susceptibility test, screening and confirmation of ESBLs were performed using the Clinical Laboratory Standard Institute's guidelines.

Results: Out of the 100 swab samples cultured, 96 (96%) showed bacterial growth. A total of one hundred and forty isolates were isolated in this study which were further identified based on cultural, morphological and biochemical characteristics. The study also found that door handles/knobs had higher level of contamination in Outpatient Departments (OPDs), Emergency, Laboratory, General wards and Toilets, in that order as compared to Radiology Room, Staff rooms, Intensive Care Unit and Operation Theatre which were lower. The level of contamination varies depending on the traffic exposure and the environment. The most prevalent Gram-negative bacteria identified was Escherichia coli 28.85%, followed by Klebsiella spp 21.15%, Pseudomonas aeruginosa 15.38%, Proteus spp 11.54%, Enterobacter spp 9.62%, Acenetobacter spp 7.69%, Citrobacter spp 5.77%. The most effective drug of choice was Amikacin, Nitrofurantoin, Norfloxacin, Ciprofloxacin, Tetracycline and Imipenem for many Gram-negative isolates. The overall prevalence of ESBLs in this study was 27.14%. Out of total 15 Escherichia coli isolated, 11(73.3%), Klebsiella spp 9/11 (81.8%); Pseudomonas spp 7/8 (87.5%), Proteus spp 4/6 (66.6%); Enterobacter spp 3/5 (60%), Acenetobacter spp 3/4 (75%) and Citrobacter spp 1/3 (33.3%) were found to be Extended β-Lactamase Producers (ESBLs).

Conclusion: The isolation of of pathogenic Gram-negative bacteria and ESBLs in hospital environments and subsequent detection of high drug resistance patterns indicates a potentially serious public health challenge that strengthens the need for the effective and routine cleaning of door-handles in hospitals.

Background: Actinomycetes are excellent microbial sources for various chemical structures like enzymes, most of which are used in pharmaceutical and industrial products. Actinomycetes are preferred sources of enzymes due to their high ability to produce extracellular enzymes. L-glutaminase has proven its essential role as a pharmaceutical agent in cancer therapy and an economic agent in the food industry. The current study aimed to screen the potent L-glutaminase producer and optimize the production media for maximum enzyme yield using one factor at a time (OFAT) approach and statistical approaches under solid-state fermentation (SSF).

Results: Out of 20 actinomycetes strains isolated from rhizosphere soil, 5 isolates produced extracellular L-glutaminase. One isolate was chosen as the most potent strain, and identified as Streptomyces pseudogriseolus ZHG20 based on 16S rRNA. The production and optimization process were carried out under SSF, after optimization using OFAT method, the enzyme production increased up to 884.61 U/gds. Further, statistical strategy, response surface methodology (RSM), and central composite design (CCD) were employed for the level optimization of significant media component (p < 0.05), i.e., wheat bran, sesame oil cake, and corn steep liquor which are leading to increase 3.21-fold L-glutaminase production as compared to unoptimized media.

Conclusions: The presented investigation reveals the optimization of various physicochemical parameters using OFAT and RSM-CCD. Statistical approaches proved to be an effective method for increasing the yield of extracellular L-glutaminase from S. pseudogriseolus ZHG20 where L-glutaminase activity increased up to 1297.87 U/gds which is 3.21-fold higher than the unoptimized medium using a mixture of two solid substrates (wheat bran and sesame oil cake) incubated at pH 7.0 for 6 days at 33 °C.

Background: Microorganisms have characteristics that aid plant growth and raise the level of vital metabolites in plants for better growth including primary and secondary metabolites as well as several developmental enzymes. Marine bacteria must endure harsh environmental circumstances for their survival so it produces several secondary metabolites to protect themselves. Such metabolites might likewise be advantageous for a plant's growth. However, the effectiveness of marine microbes on plant growth remains unexplored. In the present study, we aim to evaluate such marine microbe both in vitro and in vivo as a plant growth promoter.

Result: Marine Bacillus licheniformis was found positive for vital plant growth-promoting traits like gibberellin and ammonia production, phosphate and potassium solubilization in vitro. Due to the presence of such traits, it was able to increase germination in chickpea. As it can colonize with the roots, it will be able to help plants absorb more nutrients. Additionally, in vivo study shows that B. licheniformis treatment caused rise in vital factors involved in plant growth and development like chlorophyll, POX, phenol, proline, carotenoid, flavonoid, total proteins and SOD which resulted in increase of chickpea height by 26.23% and increase in biomass by 33.85% in pot trials.

Conclusion: Marine B. licheniformis was able to promote plant growth and increased chickpea production in both number and weight for both in vitro and in vivo conditions.

Background: Staphylococcus aureus is a gram-positive spherical bacteria and the most common cause of nosocomial infections in the world. Given its clinical significance, the genome sequence of S. aureus has been elucidated to enhance our comprehension of its lifestyle and pathogenicity. The research aimed to summarize a potential hypothetical protein that may play an important role in S. aureus virulence and pathogenicity, covering its anticipated structure, probable biological functions, and importance in this context.

Results: A hypothetical protein, YP_498675.1 with 281 amino acid residues of S. aureus, was chosen for analysis and modeling by several bioinformatics tools and databases in this work. According to primary and secondary structure analyses, YP_498675.1 is a stable hydrophilic protein with a significant proportion of α-helices. Subcellular localization predictions by CELLO, PSORTb, and SOSUI server indicate that it is a cytoplasmic protein. NCBI-CDD, Pfam, and InterProScan functional genomics research revealed that the hypothetical protein may include the pyridoxal phosphate (PLP)-dependent 2, 3-diaminopropionate biosynthesis protein SbnA domain. In the homology modeling method, the HHpred server was employed to create its 3D structure using the template structure of a Staphyloferrin B precursor biosynthetic enzyme SbnA bound to PLP (PDB ID: 5D84_A), an X-ray diffraction model having 100% sequence identity with the hypothetical protein. After energy minimization, several quality assessments and validation factors determined that the generated protein model was reliable and of reasonable quality.

Conclusion: The present study has characterized and functionally annotated the hypothetical protein YP_498675.1 of S. aureus. Further experimental validation would aid in determining the actual function of YP_498675.1 as well as confirm the protein's value as a therapeutic target.

Background: Enzymatic catalysis in different industrial applications is often preferred over chemical methods due to various advantages, such as higher specificity, greater efficiency, and less environmental footprint. Pectinases are a group of enzymes that catalyze the degradation of pectic compounds, the key components of plant middle lamella and the primary cell wall. Pectinases have found applications in multiple industrial processes, including cotton bioscouring, fruit juice extraction and its clarification, plant fiber degumming, paper making, plant biomass liquefaction, and saccharification, among others. The purpose of this study was to taxonomically characterize a bacterial species exhibiting pectinolytic activities and assess its pectinolytic activity qualitatively and quantitatively, as well as test its bioscouring potential.

Results: Here, we report that Burkholderia cepacia, a previously unknown species with pectinolytic activity, exerts such activity comparable to commercially used pectinase enzymes in the textile industry, but requires less temperature for activity.

Conclusion: Quantitative evaluation of enzyme activity indicates the potential of the bacterial species for use in the bioscouring of cotton knit fabric.