Journal, genetic engineering & biotechnology最新文献

Background: Soil metagenomics is a cultivation-independent molecular strategy for investigating and exploiting the diversity of soil microbial communities. Soil microbial diversity is essential because it is critical to sustaining soil health for agricultural productivity and protection against harmful organisms. This study aimed to perform a metagenomic analysis of the soybean endosphere (all microbial communities found in plant leaves) to reveal signatures of microbes for health and disease.

Results: The dataset is based on the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) release "microbial diversity in soybean". The quality control process rejected 21 of the evaluated sequences (0.03% of the total sequences). Dereplication determined that 68,994 sequences were artificial duplicate readings, and removed them from consideration. Ribosomal Ribonucleic acid (RNA) genes were present in 72,747 sequences that successfully passed quality control (QC). Finally, we found that hierarchical classification for taxonomic assignment was conducted using MG-RAST, and the considered dataset of the metagenome domain of bacteria (99.68%) dominated the other groups. In Eukaryotes (0.31%) and unclassified sequence 2 (0.00%) in the taxonomic classification of bacteria in the genus group, Streptomyces, Chryseobacterium, Ppaenibacillus, Bacillus, and Mitsuaria were found. We also found some biological pathways, such as CMP-KDO biosynthesis II (from D-arabinose 5-phosphate), tricarboxylic acid cycle (TCA) cycle (plant), citrate cycle (TCA cycle), fatty acid biosynthesis, and glyoxylate and dicarboxylate metabolism. Gene prediction uncovered 1,180 sequences, 15,172 of which included gene products, with the shortest sequence being 131 bases and maximum length 3829 base pairs. The gene list was additionally annotated using Integrated Microbial Genomes and Microbiomes. The annotation process yielded a total of 240 genes found in 177 bacterial strains. These gene products were found in the genome of strain 7598. Large volumes of data are generated using modern sequencing technology to sample all genes in all species present in a given complex sample.

Conclusions: These data revealed that it is a rich source of potential biomarkers for soybean plants. The results of this study will help us to understand the role of the endosphere microbiome in plant health and identify the microbial signatures of health and disease. The MG-RAST is a public resource for the automated phylogenetic and functional study of metagenomes. This is a powerful tool for investigating the diversity and function of microbial communities.

Background: Myogenic enhancer transcription factor 2A (MEF2A) is a transcription factor known for its role in controlling skeletal muscle regeneration and metabolic processes, while activating transcription factor 3 (ATF3) is a stress-induced transcription factor that plays a role in modulating metabolic processes, immunity, and oncogenesis. Environmental factors, such as dietary protein, can influence gene expression levels. Insufficient protein intake can negatively affect the metabolic performance of internal organs, leading to the abnormal weight of internal organs. A total of 192 non-sexing crossbred local chickens day-old-chick (DOC) with a completely randomized design (CRD) method of 3 treatments and 8 replicates. Real-time polymerase chain reaction (RT-PCR) is used to measure the gene expression levels. This study aimed to determine the effect of feeding with various protein levels on internal organ weight and gene expression of MEF2A and ATF3 in crossbred local chickens.

Result: The analysis of treatment revealed that the results were not significantly different (P > 0.05) on gizzard weight and spleen weight. However, it shows a significantly different result (P < 0.05) on heart weight and a highly significantly different result (P < 0.01) on pancreas weight. These findings suggest that protein levels in the diet had a significant impact on heart and pancreas weights. In terms of gene expression, the increased utilization of protein did not result in an elevation of MEF2A gene expression in both muscle tissue and liver tissue. Specifically, in muscle tissue, MEF2A gene expression was highly expressed at 18% protein feed for the starter phase and 16% for the finisher phase. Conversely, in liver tissue, MEF2A gene expression was highly expressed at 22% protein feed for the starter phase and 20% for the finisher phase. Moreover, ATF3 gene expression in muscle tissue exhibited a negative correlation with increasing feed protein levels.

Conclusion: The results indicate that varying protein levels did not lead to abnormal weights in the liver, kidney, heart, and spleen organs. Additionally, the differential gene expression patterns of MEF2A and ATF3 in muscle tissue and liver tissue suggest that these genes respond differently to varying protein-feeding treatments. These findings provide insights into the complex regulatory mechanisms of MEF2A and ATF3 genes in relation to protein levels and organ-specific responses in crossbred local chickens.

Background: Human nucleotide triphosphate diphosphatase (NUDT15) is one of the essential proteins involved in the hydrolysis of anti-cancer drugs against leukemia. Polymorphisms in NUDT15 significantly affect the hydrolysis activity that leads to side effects, including leucopenia. Drugs having a better affinity with NUDT15 protein and contributing stable conformation may benefit patients from leucopenia. Most frequent NUDT15 polymorphisms causing structure variability and their association with leukemia were screened. The selected protein variants and anti-cancer drug structures were collected. Further, molecular docking was performed between drugs and NUDT15 variants along with the wild-type. Finally, molecular dynamics were executed for 100 ns to understand the stability of the protein with the anti-cancer drug based on molecular trajectories.

Results: Three-dimensional structures of NUDT15 wild, the most frequent variants (Val18Ile, Arg139Cys, and Arg139), and the anti-cancer drugs (azathioprine, mercaptopurine, and thioguanine) were selected and retrieved from structure databases. On molecular docking the binding energies of anti-cancer drugs against NUDT15 structures ranged from - 5.0 to - 5.9 kcal/mol. Among them, azathioprine showed the highest affinities (- 7.3 kcal/mol) for the wild and variant structures. Additionally, the molecular dynamics suggest all analyzed NUDT15 were stable with azathioprine based on the dynamic trajectories.

Conclusion: Our results suggest azathioprine could be the preferable anti-cancer drug for the population with NUDT15 variants that could effectively be hydrolyzed as evidenced by molecular docking and dynamic simulation.

Background: About one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood.

Methods: We disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses.

Results: The overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells.

Conclusions: The present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis.

Background: The Pavo cristatus population, native to the Indian subcontinent, is thriving well in India. However, the Pavo muticus population, native to the tropical forests of Southeast Asia, has reduced drastically and has been categorised as an endangered group. To understand the probable genetic factors associated with the decline of P. muticus, we compared the mitogenome-encoded proteins (13 proteins) between these two species.

Results: Our data revealed that the most frequent variant between these two species was mtND1, which had an alteration in 9.57% residues, followed by mtND5 and mtATP6. We extended our study on the rest of the proteins and observed that cytochrome c oxidase subunits 1, 2, and 3 do not have any change. The 3-dimensional structure of all 13 proteins was modeled using the Phyre2 programme. Our data show that most of the proteins are alpha helical, and the variations observed in P. muticus reside on the surface of the respective proteins. The effect of variation on protein function was also predicted, and our results show that amino acid substitution in mtND1 at 14 sites could be deleterious. Similarly, destabilising changes were observed in mtND1, 2, 3, 4, 5, and 6 and mtATP6-8 due to amino acid substitution in P. muticus. Furthermore, protein disorder scores were considerably altered in mtND1, 2, and 5 of P. muticus.

Conclusions: The results presented here strongly suggest that variations in mitogenome-encoded proteins of P. cristatus and P. muticus may alter their structure and functions. Subsequently, these variations could alter energy production and may correlate with the decline in the population of P. muticus.

Background: Tubulins are highly conserved globular proteins involved in stabilization of cellular cytoskeletal microtubules during cell cycle. Different isoforms of tubulins are differentially expressed in various cell types, and their protein-protein interactions (PPIs) analysis will help in identifying the anti-microtubular drug targets for cancer and neurological disorders. Numerous web-based PPIs analysis methods are recently being used, and in this paper, I used Gene Ontology (GO) tools, e.g., Stringbase, ProteomeHD, GeneMANIA, and ShinyGO, to identify anti-microtubular target proteins by selecting strongly interacting proteins of tubulins.

Results: I used 6 different human tubulin isoforms (two from each of α-, β-, and γ-tubulin) and found several thousands of node-to-node protein interactions (highest 4956 in GeneMANIA) and selected top 10 strongly interacting node-to-node interactions with highest score, which included 7 tubulin family protein and 6 non-tubulin family proteins (total 13). Functional enrichment analysis indicated a significant role of these 13 proteins in nucleation, polymerization or depolymerization of microtubules, membrane tethering and docking, dorsal root ganglion development, mitotic cycle, and cytoskeletal organization. I found γ-tubulins (TUBG1, TUBGCP4, and TUBBGCP6) were known to contribute majorly for tubulin-associated functions followed by α-tubulin (TUBA1A) and β-tubulins (TUBB AND TUBB3). In PPI results, I found several non-tubular proteins interacting with tubulins, and six of them (HTT, DPYSL2, SKI, UNC5C, NINL, and DDX41) were found closely associated with their functions.

Conclusions: Increasing number of regulatory proteins and subpopulation of tubulin proteins are being reported with poor understanding in their association with microtubule assembly and disassembly. The functional enrichment analysis of tubulin isoforms using recent GO tools resulted in identification of γ-tubulins playing a key role in microtubule functions and observed non-tubulin family of proteins HTT, DPYSL2, SKI, UNC5C, NINL, and DDX41 strongly interacting functional proteins of tubulins. The present study yields a promising model system using GO tools to narrow down tubulin-associated proteins as a drug target in cancer, Alzheimer's, neurological disorders, etc.

Background: Colorectal Cancer (CRC) is the third most common cancer type and the second leading cause of cancer-related deaths worldwide. However, the existing treatment, as well as prognosis strategies for CRC patients, need to be improved in order to increase the chance of survival. Targeted therapies of CRC, as opposed to ordinary therapies, target key biological features and pathways of cancerous cells hence minimizing the subsequent damage to normal cells. MicroRNAs have been reported to play a crucial role in inhibiting and/or suppressing major pathways in various cancer types by targeting transcripts of key genes in such pathways.

Methods: The purpose of this study was to analyze in silico the differentially expressed genes from five microarray datasets of patients with CRC. Furthermore, miRNAs were investigated to inhibit cancer cell proliferation and metastasis by targeting a key gene-frizzled receptor 3 (FZD3) in the Wnt signaling pathway.

Results: The Wnt pathway receptor FZD3 is upregulated in CRC along with other pathway genes, which play a critical role in tumorigenesis. In contrast, miR-98-5p inhibits the activity of FZD3 by binding directly to the 3'UTR of its mRNA, therefore exerting a suppressor effect on colorectal tumors.

Conclusion: The study reveals miR-98-5p as a novel target of FZD3 and an inhibitor of the Wnt signaling pathway hence being a potential candidate for developing targeted therapies against CRC.

Background: Sheep is one of the commodities of livestock which has been known widely in Indonesia for supporting the national food security. Improvement in genetic quality by selection based on genetic markers for growth is necessary to increase meat production. Quantitative trait loci (QTL) analysis in sheep suggests that Calpain 3 gene (CAPN3) gene might be one of the candidate loci affecting growth traits. CAPN3 is located on chromosome 7 sheep expressed in the skeletal muscles. The aim of this study was to investigate polymorphism CAPN3 intron 11 in Merino × Garut (MEGA) backcross using the PCR-RLFP method and to determine their association with growth traits.

Results: SNP intron 11 CAPN3 | BseSI of Merino × Garut (MEGA) backcross sheep was polymorphic and resulted in two alleles of C and T with a frequency of 0.76 and 0.24, respectively, and CC, CT, and TT genotypes with a frequency of 0.54, 0.43, and 0.02, respectively. These loci were found to be in Hardy-Weinberg equilibrium. The SNP CAPN3 | BseSI significantly affected (P < 0.05) the birth weight in Merino × Garut (MEGA) backcross sheep.

Conclusion: This result suggests that the CAPN3 | BseSI can be used as a genetic marker for birth weight trait in sheep.

Background: Exopolysaccharides are extremely powerful molecules with a wide range of uses in pharmaceuticals due to their structural and compositional complexity. Marine microorganisms often produce bioactive substances with novel functions and structures because of their special living conditions. Polysaccharides from marine microorganisms are of interest to new drug discovery.

Results: The current research focused on the isolation of bacteria from Red Sea, Egypt, that have the ability to produce a new natural exopolysaccharide in order to be examined in treating Alzheimer's illness to obviate side effects of synthetic drugs. Properties of exopolysaccharide (EPS) produced by an isolated Streptomyces strain were investigated for its capability to play as anti-Alzheimer. This strain was identified morphologically, physiologically, and biochemically and actually was confirmed by molecularly 16S rRNA analysis as Streptomyces sp. NRCG4 with accession number MK850242. The produced EPS was fractionated by precipitation 1:4 volumes of chilled ethanol and the third major fraction (1:3) listed as NRCG4, and then the functional groups, MW, and chemical evaluation have been detected via Fourier-transform infrared (FTIR), high-performance gel permeation chromatography (HPGPC), and high-performance liquid chromatography (HPLC). The findings showed that NRCG4 was an acidic EPS composed of mannuronic acid, glucose, mannose, and rhamnose in a molar ratio of 1.2:1.5:2.8:1.0, respectively. NRCG4 Mw was determined to be 4.25 × 10⁵ gmol^-1 and the Mn to be 1.97 × 10⁵ gmol^-1. Also, the NRCG4 included uronic acid (16.0%) and sulfate (0.0%), but no protein was found. In addition, antioxidant and anti-inflammation activity was measured through various methods. This study confirmed that NRCG4 exopolysaccharide exerted anti-Alzheimer's characters via inhibition of cholinesterase and tyrosinase as well as anti-inflammatory and antioxidant abilities. Additionally, it occurred a potential role in the suppression of Alzheimer's disease risk factors through its antioxidant (metal chelation, radical scavenging capability), anti-tyrosinase and anti-inflammatory characteristics. The anti-Alzheimer's disease efficacy of NRCG4 exopolysaccharide may be assigned to its unique determined chemical composition.

Conclusions: The present study highlighted those exopolysaccharides could be harnessed to improve pharmaceutical industry (anti-Alzheimer, anti-tyrosinase, anti-inflammatory, and antioxidant agents).

Introduction: Cancer is a major issue in medical science with increasing death cases every year worldwide. Therefore, searching for alternatives and nonorthodox methods of treatments with high efficiency, selectivity and less toxicity is the main goal in fighting cancer. Acetyl-11-keto-β-boswellic acid (AKBA), is a derivative pentacyclic triterpenoid that exhibited various biological activities with potential anti-tumoral agents. In this research, AKBA was utilized to examine the potential cytotoxic activity against MCF-7 cells in vitro and monitor the cellular and morphological changes with a prospective impact on apoptosis induction.

Methods: The cytotoxic activity of AKBA was measured by 3(4,5dimethylthiazole- 2-yl)-2,5 diphyneltetrazolium bromide (MTT) assay. A dose-dependent inhibition in MCF-7 cell viability was detected. The clonogenicity of MCF-7 cells was significantly suppressed by AKBA increment in comparison with untreated cells.

Result: Morphologically, exposure of MCF-7 cells to high AKBA concentrations caused changes in cell nuclear morphology which was indicated by increasing in nuclear size and cell permeability intensity. The mitochondrial membrane potential (ΔΨm) was reduced by increasing AKBA concentration with a significant release of cytochrome c. Acridine orange/ethidium bromide dual staining experiment confirmed that MCF-7 cells treated with AKBA (IC50 concentration) displayed a late stage of apoptosis indicated by intense and bright reddish colour.

Conclusion: A significant increase in reactive oxygen species formation was observed. Caspase 8 and caspase 9 activities were estimated and AKBA activated the production of caspase 8 and caspase 9 in a dose-dependent pattern. Finally, the cell phase distribution analysis was conducted, and flow cytometric analysis showed that AKBA at 200 μg mL-1 significantly arrest MCF-7 cells at the G1 phase and triggered apoptosis.