Biochemical Genetics最新文献

Cranial defect repair remains a clinical challenge in current surgical practice. Matrix extracellular phosphoglycoprotein (MEPE) plays a role in mineralization, but its specific mechanism in calvarial bone repair, particularly concerning oxidative stress, remains unclear. Bioinformatics analysis identified hub genes and pathways from the calvarial defect dataset GSE20980 using differentially expressed genes screening (p < 0.05, |log2Foldchange|≥3) and weighted gene co-expression network analysis. In vivo, a rat critical-sized calvarial defect (CSD) model was established. MEPE was knocked down via lentiviral siRNA delivery. Bone repair was assessed 8 weeks post-surgery using micro-computed tomography (bone volume/total volume [BV/TV] and trabecular number [Tb.N]), histology (hematoxylin-eosin staining and tartrate-resistant acid phosphatase staining). Enzyme-linked immunosorbent assay was performed to evaluate levels of inflammatory cytokines and oxidative stress indicators. MEPE-associated pathways were screened, and the protein levels were measured by western blot. Bioinformatics analyses identified MEPE as a key upregulated hub gene in bone defects. In vivo, MEPE expression was significantly elevated in CSD rats. MEPE knockdown enhanced bone repair (increased BV/TV and Tb.N), and promoted bone formation and angiogenesis. Furthermore, knockdown of MEPE reduced inflammation (decreased tumor necrosis factor alpha, interleukin 6, interleukin-1β), reactive oxygen species level, increased antioxidants (elevated catalase, glutathione, superoxide dismutase) and upregulated osteogenic markers (runt-related transcription factor 2, osteocalcin, alkaline phosphatase). Bioinformatics analysis of intersecting genes implicated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway as the downstream pathway of MEPE. Mechanistically, MEPE knockdown reversed CSD-induced suppression of cAMP and PKA protein expression. MEPE knockdown promotes calvarial bone repair by mitigating oxidative stress, reducing inflammation, enhancing osteogenesis, and activating the cAMP/PKA signaling pathway, representing a potential therapeutic target for bone regeneration.

Colorectal cancer (CRC) is a relatively widespread malignancy that contributes to considerable mortality and healthcare challenges. Recent studies emphasize the significance of N6-methyladenosine (m⁶A) RNA modification in CRC progression. However, research on KIAA1429 (a key m⁶A methyltransferase) is still limited during CRC. This study focuses on investigating the impact of KIAA1429-driven m⁶A modification on CRC progression. The expression of KIAA1429 in CRC was assessed via bioinformatics analyses and qRT-PCR methods. Its impact on CRC cell malignancy was examined by employing CCK8, colony formation, wound healing, and transwell invasion assays. The relationship among KIAA1429 and Family with sequence similarity 84, member B (FAM84B) was verified via qRT-PCR, immunoblotting, and MeRIP. Finally, the effect of their interaction on CRC cell malignancy and Wnt/β-catenin signaling was assessed in vitro and in vivo. The KIAA1429 expression was markedly high in CRC, and silencing it significantly reduced malignant phenotypes of CRC cells. The bioinformatics analysis identified FAM84B as a target gene of KIAA1429 in CRC, with elevated expression and m⁶A-dependent methylation regulation by KIAA1429. The outcomes of qRT-PCR, immunoblotting and MeRIP assay confirmed a positive association between KIAA1429 and FAM84B. Furthermore, KIAA1429 silencing partially decreased β-catenin levels and reversed the malignant effects of FAM84B overexpression on CRC cells, both in vitro and in vivo. The results illustrate that KIAA1429 promotes CRC tumorigenesis by stabilizing FAM84B mRNA and activating the Wnt/β-catenin pathway, highlighting its capability as a prognostic biomarker and therapeutic target for CRC.

The procurement of blood and tissue samples for DNA extraction in avian species intended for molecular studies is associated with the induction of discomfort and pain in the subjects, compounded by practical challenges in application and ethical considerations. Consequently, feathers have emerged as a more prevalent source for molecular investigations, particularly in the fields of poultry and ornithology. However, the effective extraction of DNA from feathers necessitates the breakdown of the hard keratinized tissue within the feather structure. This study aimed to devise a highly efficient, cost-effective, and easily adaptable Modified Phenol-Chloroform (MPC) approach for genomic DNA extraction from feathers, addressing shortcomings identified in previous studies on feather-based DNA isolation. The MPC method was employed to extract genomic DNA from feather samples obtained from six distinct avian species (chicken, guinea fowl, canary, pigeon, emu, and goose). Comparative evaluation of DNA isolation efficiency was conducted by employing two different commercial DNA kits alongside the MPC method. The results showed significantly higher DNA concentrations (ng/ml) from chicken feathers using the MPC method compared to those obtained with commercial kits (p < 0.05), along with high DNA purity (1.83 ± 0.11). Subsequent PCR experiments, employing nuclear and mitochondrial DNA-specific primers, illustrated the effective amplification of short and long fragments from MPC-isolated DNA samples. In contrast to commercial kits, the findings underscore the successful application of the MPC method in isolating high-quality genomic DNA from feathers characterized by elevated keratin content.

Eucalyptus is a fast growing hardwood tree species preferred for its wood properties which are suitable for paper and pulp industries. The transcriptional regulation governing secondary wood formation in Eucalyptus is extensively studied while the role of post-transcriptional mechanism determining wood phenotypes is not well documented. The present study aimed at understanding the miRNA-mediated regulation of secondary development in Eucalyptus tereticornis. Transcriptome-wide identification of miRNAs in wood tissues predicted a total of 266 conserved mature miRNA members belonging to 76 families. Et-miR156 was the most abundant family followed by Et-miR166 and Et-miR167. Majority of the gene targets of miRNAs were transcription factors including AP2, GRF, TCP, ARF, bHLH, bZIP, HD-ZIP, MYB, NAC, SBP, WRKY and Zinc finger. Further, 102 miRNA members were predicted to target genes from the cellulose and lignin biosynthetic pathways. Additionally, the expression patterns of four miRNAs (Et-miR156d-5p, Et-miR156a, Et-miR156b-5p, and Et-miR159b) and their gene targets were validated in eight individuals with contrasting cellulose and lignin content to predict the role of miRNAs in governing wood property traits. In summary, the present study has predicted potential miRNA targets which may regulate wood phenotypes in E. tereticornis and has also generated valuable genomic resource for enhancing wood quality through marker assisted selection and gene editing strategies.

As a multifactorial and endocrine disease, polycystic ovary syndrome (PCOS) affects approximately 5-20% of women worldwide. Recently, long noncoding RNAs (lncRNAs) have emerged as potent predictors of a particular phenotype in PCOS. Our preliminary study examines the link between polymorphisms in lncRNAs MEG3, HOTTIP, and GAS5 and the risk of PCOS. The present study included 200 women with PCOS and 200 healthy women. The studied variations were genotyped by applying the PCR-RFLP and the tetra-ARMS-PCR reaction) techniques. The effect of variation in lncRNA on miRNA:lncRNA interactions, lncRNA-RNA interaction network, and the impact of the variations on the splicing site were predicted using different computational databases. The codominant heterozygous (TC vs. TT) model, the dominant (TC + CC vs. TT) model, the overdominant (TT + CC vs. TC) model, the C allele of rs2023843, and the C allele of rs55829688 had a protective role against PCOS. The A allele of rs4081134 and G allele of rs7158663 of the MEG3 conferred an increased risk of PCOS by 1.37 and 1.44 folds, respectively. The interaction analysis revealed that TC/GG/AA/TC and TC/GG/GA/TC strongly decreased the risk of PCOS by 94 and 92%, respectively. Interestingly, MEG3 and HOTTIP variants can create or disrupt binding sites for several splicing factors. In our population, MEG3 rs4081134 and rs7158663, GAS5 rs55829688, and HOTTIP rs2023843 polymorphisms were associated with PCOS risk. Replication studies on larger sample sizes must be conducted to confirm these findings and investigate other potential causative factors involved in the pathophysiology of PCOS.

Black turmeric (Curcuma caesia Roxb.) is widely used in traditional medicine due to its antifungal, antibacterial, anticancer, analgesic, anti-inflammatory, and anticonvulsant properties. Despite these properties, systematic phytochemical evaluation of different genotypes remains limited. This study investigated the biochemical diversity of 21 genotypes collected from different agro-climatic regions of India and cultivated under a randomized block design at two distinct environments- Bhavanisagar and Coimbatore, Tamil Nadu, India. Methanolic extracts of rhizomes were quantitatively assessed for total phenolics, flavonoids, proteins, alkaloids, and cardiac glycosides. The analysis revealed significant variation attributable to both genotypic differences and environmental conditions. At Bhavanisagar, genotype GCA-5 recorded the highest phenolic (3.81 mg GAE/g DW) and cardiac glycoside (183.38 mg SE/g) contents, whereas GMN-10 exhibited the maximum flavonoid concentration (38.29 mg QE/g). The highest protein content was observed in GAN-16 (165.86 mg/100 g DW), while GTE-18 showed the greatest alkaloid accumulation (227.34 mg AE/g). Notably, GTE-18 also ranked among the top performers across most traits at Coimbatore, indicating its strong stability across environments. Correlation analysis revealed a significant positive relationship between cardiac glycosides and phenols (r = 0.49**), proteins (r = 0.40**), and flavonoids (r = 0.34**), as well as between flavonoids and antioxidant capacity (r = 0.59**) and proteins (r = 0.22**), suggesting coordinated biosynthesis. These interrelationships highlight potential metabolic linkages and support the pharmacological synergy of secondary metabolites. Genotypes GCA-5, GTE-18, and GMN-10 demonstrated superior phytochemical accumulation and adaptability across environments, indicating their potential for pharmaceutical and nutraceutical applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a highly efficient genome editing tool that can genetically disrupt genes and genetic elements, making it a timely, cost-effective, and powerful tool for studying gene function. The success of gene editing depends on the ability to introduce CRISPR components, including guide RNA (gRNA) and Cas9 nuclease, into the target cell, which is challenging in numerous difficult-to-transfect cell types, such as cardiomyocytes. Lentiviral vectors (LVs) are among the primary delivery methods for the CRISPR/Cas9 system as they can stably maintain robust expression in various dividing and non-dividing cells. However, stably integrated LVs consistently express CRISPR/Cas9 components at high levels, rendering them susceptible to off-target effects. New-generation integrase-deficient LV (IDLV) offers an attractive alternative approach for delivering CRISPR/Cas9 components. This study constructed transient receptor potential cation channel mucolipin subfamily member 1 gene knockout models in H9C2 cell lines using IDLVs. Strategies for gRNA design and screening, the IDLV packaging process, CRISPR delivery, and knockout validation are outlined. These protocols will assist researchers in the application of CRISPR technology to study gene function in mammalian cells.