Molecular Biology Research Communications最新文献

Acute myeloid leukemia (AML) is the most frequent type of leukemia among adults. Investigating AML heterogeneity based on DNA methylation can improve clinical diagnosis and prognosis. This study was conducted to investigate NR4A1 and NR4A3 gene methylation in fifty newly diagnosed AML patients and fifty healthy controls using Methyl specific PCR (MSP). The frequency of the rs1569686 in the DNMT3B was also determined by Tetra primer ARMS PCR. Also, the association between methylation of studied genes and some prognostic marker including mutation of FLT3 and NPM genes, as well as some hematological factors of patients was evaluated. According to the findings, AML patients have a significantly higher prevalence of methylated NR4A1 and NR4A3 genes than those without AML. AML patients with un-methylated NR4A3 had significantly higher frequency of FLT-ITD positivity than AML patients with methylated NR4A3. Also, there was no significant association between rs1569686 and AML. Finally, the distribution of different genotypes of rs1569686 between AML patients with and without methylation in NR4A1 and NR4A3 did not show any significant association. The results found that NR4A1 and NR4A3 were hyper-methylated in AML patients. However, rs1569686 polymorphism was not a main contributor to methylation status of studied gene. Future studies should consider other mechanisms influencing the role of NR4A1 and NR4A3 hypermethylation in AML.

Single nucleotide polymorphisms (SNPs) are pivotal in understanding the genetic basis of complex disorders. Among them, nonsynonymous SNPs (nsSNPs) that alter amino acid sequences can significantly impact protein structure and function. This study focuses on analyzing deleterious nsSNPs in the tumor suppressor gene PTEN (Phosphatase and TENsin Homolog), which plays a central role in regulating the PI3K/Akt signaling pathway and tumorigenesis. Out of 43,855 SNPs in PTEN, 17 deleterious nsSNPs were identified using six computational tools. Protein stability analysis revealed that 15 variants reduce stability, potentially leading to functional impairment. Structural evaluations using HOPE and ConSurf classified mutations into buried structural residues disrupting protein integrity and exposed functional residues affecting molecular interactions. STRING database analysis highlighted PTEN as a central node in an intricate protein network, with deleterious mutations impairing critical interactions with partners such as PIK3CA, AKT1, and TP53. Secondary structure analysis revealed distinct structural deviations, particularly for G129E, which exhibited the most pronounced destabilization. Molecular dynamics simulations confirmed stability variations across mutants, with G129E exhibiting greater instability. This comprehensive analysis enhances understanding of PTEN nsSNP impacts, offering insights for therapeutic interventions and future experimental validation.

Colony Stimulating Factor-1 Receptor (CSF1R) is a tyrosine kinase transmembrane receptor that plays a vital role in innate immunity and neurogenesis and controls the differentiation and maintenance of most tissue-resident macrophages. CSF1R mutations have been linked with many neurodegenerative diseases. In this work, we aim to identify the functional and structural impact of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) mutations on CSF1R, which could help understand the consequences of these mutational changes. A consensus-based prediction approach was used to screen the missense SNPs using six in-silico tools: SIFT, PROVEAN, PMut, MutPred, MISSENSE 3D, and FATHMM. SNPs found to be deleterious by more than five out of six tools were subjected to further analysis, such as protein secondary structure and domain architecture analysis by PSIPRED and NCBI-CDD, respectively. Mutant models of highly deleterious SNPs were modeled using PyMol, followed by energy minimization and Root Mean Square Deviation (RMSD) analysis and molecular dynamic (MD) simulation by YASARA, TM-ALIGN, and WebGro, respectively. Out of 780 missense SNPs screened, we found the four most deleterious SNPs (L301S, A770P, I775N, and F849S) that decreased the protein stability because of their presence in the conserved regions of wild-type CSF1R. Structural and functional studies revealed that these mutations could disrupt the protein's core and surface interactions, leading to destabilization and functional impairment. Moreover, the mutated proteins exhibited enhanced conformational flexibility and instability, as confirmed by MD simulation analysis.

Colorectal cancer (CRC), which has high mortality and increasing morbidity is a major concern worldwide. The autophagy pathway plays a crucial role in carcinogenesis and drug resistance in this disease. Epigenetic modification is one of the main regulatory mechanisms for this pathway. This study aimed to investigate the impact of promoter methylation as one of the epigenetic modifications on the expression of autophagy-associated genes (ATGs) (ATG2B, ATG4D, ATG9A, and ATG9B) in 21 CRC patients from southern Iran. The tissue DNA and RNA were extracted by standard phenol-chloroform extraction method and A BIOZOL RNA isolation kit, respectively. The methylation status and transcript levels of desired genes were ascertained using the methylation-specific PCR and quantitative real-time PCR methods, respectively. In the majority of studied patients, the relative mRNA expressions of ATGs were significantly higher in CRC tissues compared to normal ones. There was no significant relationship between the methylation of the ATG genes and clinicopathological features of CRC patients. Interestingly, in most of the patients, the promoter hypermethylation of the ATG2B, ATG4D, ATG9A and ATG9B genes led to their high mRNA expression. Although promoter hypermethylation usually suppresses gene expression, the cancer type, stage, and compensatory mechanisms may reverse this association. This highlights the complexity of the epigenetic regulation of ATG2B, ATG4D, ATG9A and ATG9B genes in CRC. Further large-scale studies will contribute to discovering the exact influences of ATG methylation in CRC carcinogenesis and thereby may thereby provide novel targets and biomarkers for this lethal illness.

Chinese Hamster Ovary (CHO) cells are essential in biopharmaceutical manufacturing. Scientists use CRISPR to enhance productivity. mRNAs contain UTRs that regulate gene expression, affecting protein abundance. Targeting these regions creates desirable knockout cells. The Caspase 8 Associated Protein 2 (CASP8AP2) gene is a promising target for improving host cell viability. This study used the CRISPR-Homology-Independent Targeted Integration (HITI) strategy to modify the 3'UTR region of the CASP8AP2 gene in CHO cells. The aim was to evaluate the effects of CASP8AP2 silencing on cell proliferation, viability, apoptosis, and the cell cycle. CASP8AP2 silencing was assessed post-modification by extracting genomic DNA from modified and unmodified CHO cells, followed by PCR and sequencing to confirm deletions. Cell proliferation and viability were measured using MTT assays, and cell cycle analysis was performed via flow cytometry. Apoptosis was evaluated through Annexin V/PE staining and flow cytometry, with apoptosis resistance assessed by determining the IC₅₀ of sodium butyrate. Results showed CASP8AP2 deletion did not affect cell proliferation or the cell cycle but improved CHO cell viability and increased resistance to apoptosis. The IC₅₀ for sodium butyrate was higher in CASP8AP2 knockout cells (7.84 mM) compared to native cells (3.43 mM), indicating enhanced apoptosis resistance. This study highlights CASP8AP2's role in apoptosis regulation without impacting cell proliferation or the cell cycle. CASP8AP2 deletion enhances viability and resistance to apoptosis, suggesting it as a target for improving recombinant protein production. Further research is needed to elucidate the molecular mechanisms and develop therapeutic strategies based on this approach.

Testis specific gene antigen 10 (TSGA10) is a protein which has roles in spermatogenesis and cancers so that deletion or mutation in the TSGA10 gene resulted in non-obstructive infertility and aberrant expression of this protein, was detected in solid tumors and leukemia. Despite the crucial roles of TSGA10 in tumorigenesis and infertility, yet it is not obvious how various nsSNPs of its gene impress the structure and function of the TSGA10. Therefore, it is worthwhile to investigate the potential highly deleterious nsSNPs by several in-silico tools before launching costly experimental approaches. In the current study, we employed several different machine learning algorithms in a two-step screening procedure to analyze single nucleotide substitutions of TSGA10 gene. Prediction tools were included SIFT, PROVEAN, PolyPhen-2, SNAP2, SNPs & GO, PhD-SNP for the first step and the second step included predictive tools such as I-mutant 3.0, MUpro, SNPeffect 4.0 (LIMBO, WALTZ, TANGO, FoldX), MutationTaster and CADD. Also, the 3D models of significantly damaging variants were built by Phyre2. The results elucidated 15 amino acid alterations as the most deleterious ones. Among these S563P, E578K, Q580P, R638L, R638C, R638G, R638S, L648R, R649C, R649H were located in a domain which is approved to has interaction with the HIF1-A protein and D62Y, R105G, D106V and D111Y were located on phosphodiesterase domain. In sum, these predicted mutations significantly influence the function of TSGA10 and they could be used for precise study of this protein in infertility and cancer experimental investigations.

The bioremediation method is considered an economical and environmentally friendly strategy for the remediation of oil-contaminated soils. However, some oil field areas have extreme environmental conditions that make it difficult to establish microbes for bioreme-diation. In this study, bacteria were isolated from oil-contaminated soils of the Dehloran oil fields, which have very harsh soil and weather conditions. Soil samples were collected from two highly contaminated mud pits. The petroleum content and physicochemical characteristics of the soil were investigated. Soil samples pollution were about 8%, sandy and alkaline, and their EC reached up to 125.6 ds/m in some samples. The isolated bacteria were screened according to their ability to grow on the M9 mineral medium containing crude oil as the sole carbon source. Moreover, their physiological characteristics in diesel degradation were investigated. The phenotypic, biochemical, and molecular characteristics of selected isolates and their stability under extreme conditions such as drought, salinity and high temperatures were investigated. Two isolates NC39 and NB391 showed the highest ability in diesel degradation. The results of 16SrRNA sequencing showed that NC39 isolate had 98% similarity to Pseudomonas sp. and isolate NB391 belonged to Pantoea agglomerans with 99% similarity. These two isolates showed a high ability to tolerate high salinity (10%), temperature (50°C), and drought (-0.73 MPa) stress. Exploiting these extremophile strains is a promising tool in the bioremediation of oil-contaminated soils in extreme environments.

Previously, the sequence of the HIV-1 reverse transcriptase gene was analyzed to identify mutations associated with drug resistance. We statistically analyzed the relationship between a set of additional data and increasing mutations. Existing sequences were also phylogenetically analyzed. Of all patients tested for phylogenetic tree analysis, one individual had the F subtype, two had the CRF01-AE strain, and two had the A subtype. Phylogenetic tree analysis revealed that HIV-1 CRF35-AD was the most prevalent subtype (88.6%) among the cases studied. The number of treatment discontinuations (r=0.621, df=20, p=0.002) and the duration of treatment (r=0.452, df=20, p=0.035) were significantly correlated with an increase in mutations.

Circular RNAs (circRNAs) are non-coding, single-stranded RNAs considered by their closed-loop structures. Research has established a connection between circRNAs and cancer progression. The objective of this project was to evaluate the expression levels of a newly discovered circRNA, circRFX3 (hsa_circRFX3_003), along with its target gene, miR-587. The study involved 60 patients diagnosed with Colorectal cancer (CRC) and 60 healthy individuals as controls. Total RNA was extracted from blood samples, converted into cDNA, and analyzed using qRT-PCR. The findings revealed an up-regulation of miR-587 and a down-regulation of circRFX3 in the blood samples of CRC patients. An inverse relationship was observed between the levels of miR-587 and circRFX3; however, there was no significant difference in circRFX3 expression levels between stages I+II and stages III+IV. The levels of miR-587 expression were linked to tumor size and location. Both circRFX3 and miR-587 play significant roles in the pathophysiology of CRC; however, additional research is necessary to elucidate their specific contributions to CRC development.

The Type V secretion system, or "autotransporter", is a secretion system that enables bacteria to directly export proteins from the cell interior to the extracellular membrane. mCherry is a second-generation monomeric red fluorescent protein that has an improvement in photostability compared to the first generation of RFP. In this research, we conducted the fusion of the mRFP into the C-terminal domain of EhaA - the translocation domain of the autotransporter protein transport system - to investigate the expression of mRFP on the surface of Escherichia coli , a model organism commonly utilized in recombinant protein research. The induction of the mRFP-EhaA C-terminal domain complex expression was achieved using isopropyl β-D-1-thiogalactopyranoside (IPTG) and confirmed through SDS-PAGE stained with Coomassie Brilliant Blue and Western blotting using anti-6X His tag antibodies. The surface expression of the mRFP-EhaA C-terminal complex protein was validated through the fluorescent properties of mRFP and further confirmed using fluorescent microscopy. This study laid the groundwork for surface expression on cost-effective Gram-negative bacteria, E. coli.