Efficient and safe extraction of microRNAs (miRNAs) from biological samples is pivotal for genetic regulation studies and biotechnological applications. This study focuses on optimizing the microRNA extraction process from the plasma of common carp, a significant species in aquaculture. Recognizing the limitations and hazards of commercial extraction kits, which often employ toxic chemicals like phenol and chloroform, we sought to develop a safer and more effective alternative. Our optimized protocol utilizes guanidinium isothiocyanate (GITC) and sarkosyl, omitting hazardous substances. We explored several parameters including GITC concentration, the addition of sarkosyl, and the role of sodium chloride in enhancing miRNA yield. Our findings demonstrate that optimal conditions involve a GITC concentration of 4.2 M, a 3% sarkosyl concentration, and the use of sodium chloride at 0.5 M. We also investigated the utility of glycogen as a nucleic acid carrier, finding 160 µg to be the optimal concentration. Comparative analysis with commercial kits indicated our method provides higher miRNA yields with reduced cycle threshold values, underscoring the effectiveness of our custom protocol. This optimized approach not only enhances miRNA recovery but also emphasizes safety and cost-effectiveness, making it a valuable method for both research and practical applications in aquaculture.
Background: This study aimed to investigate the association of rs12976445 polymorphism in the promoter region of miR-125a and rs2114358 in the precursor region of miR-1206 to breast cancer susceptibility.
Method: A total of 230 participants (110 breast cancer and 120 controls) enrolled in this study and extracted genomic DNA. The genotypes were determined by the Tetra-ARMS method. The allele and genotype frequencies were determined.
Results: Allele variation in the rs12976445 (miR-125a) sequence increased the risk of breast cancer; a significant relationship was observed between breast cancer and allele change in individuals with the C allele (p = 0.01). However, allele variation in the rs2114358 (miR-1206) decreased the risk of breast cancer in individuals with allele A (p = 0.01). In silico study showed that allele change was associated with a reduction in structural stability.
Conclusion: Therefore, the rs12976445 variant can be considered a risk factor for breast cancer, and the rs2114358 variant is a protective factor against it.
Obesity is a common public health problem associated with serious, life-threatening complications. MicroRNAs (miRs) have modulating roles in the immune and inflammatory systems. Therefore, this study aimed to analyze the relationship between miR-146a and morbid obesity (MO) in a Turkish population. In this study, a total of 258 subjects (110 patients with MO and 148 controls) were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyze miR-146a rs2910164. Then, we examined the patients as males and females separately. The results of the analyses were evaluated for statistical significance. There was a significant difference in genotype and allele frequencies of miR-146a rs2910164 between patients with MO and control individuals. miR-146a rs2910164 CC genotype and C allele were shown to increase in the MO patients' group compared to the control group (p = 0.000, p = 0.000, respectively). Also, the C allele was higher in both female and male patients compared to controls (p = 0.000, p = 0.000, respectively). High differences were also observed when the patients and the controls were compared according to CC versus GG + GC and GG versus GC + CC (p = 0.000, p = 0.000, respectively). A significant difference was found between the female/male patients and the female/male controls in terms of GG + GC versus CC (p = 0.000, p = 0.000, respectively). To the best of our knowledge, this is the first study to investigate the relationship between this variant and MO in Turkey. Our results showed that miR-146a rs2910164 is a valuable biomarker that can be used to distinguish between patients with MO and the healthy population. The findings can be extended by increasing the sample sizes with diverse ethnicities.
Several studies have reported the relationship between LIN28A gene polymorphisms (rs3811463 T > C and rs34787247 G > A) and cancer susceptibility, but the results are inconsistent and need further clarification. The current study aimed to evaluate their relationship and also to explore the relationship between LIN28A gene expression and immune infiltration, tumor stage, survival prognosis, and drug sensitivity in pan-cancer. The meta-analysis and data mining were completed by STATA software and the GSCA platform, respectively. The meta-analysis showed that the rs3811463 polymorphism was not associated with cancer susceptibility, while the rs34787247 polymorphism was associated with cancer susceptibility in the Chinese population [AA vs. GG: Odd Ratio (OR)=1.98, 95% Confidence Interval (CI)=1.35-2.89, PZ<0.001; GA vs. GG: OR = 1.17, 95%CI= 1.01-1.36, PZ=0.04; (AA + GA) vs. GG: OR = 1.24, 95%CI = 1.07-1.43, PZ=0.004; AA vs. (GA + GG): OR = 1.90, 95%CI = 1.30- 2.78, PZ=0.001; A vs. G: OR = 1.27, 95%CI = 1.12-1.44, PZ<0.001]. LIN28A gene expression was associated not only with immune infiltration, pathological stage, and survival prognosis of certain cancers, but also with sensitivity to multiple anticancer drugs, such as cisplatin, pazopanib, olaparib, and selumetinib. In conclusion, the current study suggested that the rs34787247 G > A polymorphism might be used as a cancer risk marker in the Chinese population, and LIN28A might serve as a prognostic marker and therapeutic target for certain cancers.
In genomic research, identifying the exon regions in eukaryotes is the most cumbersome task. This article introduces a new promising model-independent method based on short-time discrete Fourier transform (ST-DFT) and fine-tuned variational mode decomposition (FTVMD) for identifying exon regions. The proposed method uses the N/3 periodicity property of the eukaryotic genes to detect the exon regions using the ST-DFT. However, background noise is present in the spectrum of ST-DFT since the sliding rectangular window produces spectral leakage. To overcome this, FTVMD is proposed in this work. VMD is more resilient to noise and sampling errors than other decomposition techniques because it utilizes the generalization of the Wiener filter into several adaptive bands. The performance of VMD is affected due to the improper selection of the penalty factor (α), and the number of modes (K). Therefore, in fine-tuned VMD, the parameters of VMD (K and α) are optimized by maximum kurtosis value. The main objective of this article is to enhance the accuracy in the identification of exon regions in a DNA sequence. At last, a comparative study demonstrates that the proposed technique is superior to its counterparts.
The pharmacokinetics and tissue distribution of renadirsen sodium, a dystrophin exon-skipping phosphorothioate-modified antisense oligonucleotide with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), after subcutaneous or intravenous administration to cynomolgus monkeys were investigated. The plasma concentration of renadirsen after subcutaneous administration at 1, 3, and 10 mg/kg increased with the dose. The absolute bioavailability at 3 mg/kg after subcutaneous administration was calculated as 88.6%, and the time to reach maximum plasma concentration of renadirsen was within 4 h, indicating the efficient and rapid absorption following subcutaneous administration. The exposure of muscle tissues to renadirsen was found to increase with repeated dosing at 6 mg/kg, and higher exposure was observed in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen achieved more exon 45-skipped dystrophin mRNA in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen also showed a cumulative skipping effect in a repeated-dose study. The findings on exon 45-skipped dystrophin mRNA in these muscle tissues were consistent with the concentration of renadirsen in these tissues. Because it is not feasible to directly evaluate drug concentration and exon skipping in the heart and diaphragm in humans, the pharmacokinetics and pharmacodynamics of renadirsen in these tissues in monkeys are crucial for the design and interpretation of clinical settings.
Bladder urothelial carcinoma (BLCA), a prevalent malignant neoplasm affecting the human urinary system, is frequently linked with an unfavorable prognosis in a significant proportion of individuals. More effective and sensitive markers are needed to provide a reference for prognostic judgment. We obtained RNA sequencing data and clinical information of individuals from TCGA, and 133 copper metabolism-related genes from literature. Prognostic genes were evaluated by univariate/multivariate Cox regression analysis and LASSO analysis, and a risk-scoring model was established and validated in the GEO dataset. The CIBERSORT method was utilized to explore immune cell infiltration in BLCA individuals. In addition, tumor immune dysfunction and exclusion (TIDE) and immunophenoscore (IPS) were utilized to verify whether the model can foretell the response of BLCA individuals to immunotherapy. We successfully constructed an 8-gene risk scoring model to foretell individuals' overall survival, and the model performed well in TCGA training and GEO validation cohorts. Lastly, a nomogram containing clinical parameters and risk scores was constructed to help individualized result prediction for individuals. Calibration curves demonstrated a high degree of concordance between the observed and projected survival durations, attesting to its exceptional predictive accuracy. Analysis utilizing CIBERSORT unveiled elevated levels of immune cell infiltration in individuals classified as low risk. TIDE and IPS analyses substantiated that low-risk individuals exhibited a more favorable response to immunotherapy. In summary, the model held immense potential for stratifying the risk of survival and guiding tailored treatment approaches for individuals with BLCA, thereby offering valuable insights for personalized therapeutic interventions.
A few interactions should be considered during the detritylation reaction of solid-phase oligonucleotide synthesis (SPOS): (i) interaction of solvent with acid; (ii) interaction (or reaction) of solvent with trityl cation, and (iii) interaction of scavenger with acid, with the last one as the focus of this work. Using a stopped-flow setup, commonly used trityl cation scavengers (methanol, thioanisole, 1-dodecanethiol, triisopropylsilane, triethylsilane, and trihexylsilane) were evaluated for their reactivity toward tritylium hexafluorophosphate. Among the scavengers screened, methanol and thioanisole were found to be the most and least reactive, respectively; however, methanol does interact and react with trichloroacetic acid, thus it should not be pre-mixed and stored with acid as deblock solutions. Overall, all aspects of interactions must be taken into consideration while optimizing the detritylation reaction, especially for large scale SPOS.
Objective: The study aimed to elucidate the role and the underlying mechanism of human epididymis protein 4 (HE4) in the pathogenesis of hyperoxia-induced bronchial dysplasia in newborn rats.
Methods: Forty neonatal Sprague-Dawley (SD) rats were separated into two groups: a normal control group (20.8% oxygen concentration) and a hyperoxia-induced group (85% oxygen concentration). Three time intervals of 24 h, 3 days and 7 days were chosen for each group. Haematoxylin-eosin staining was used to identify the pathological alterations in the lung tissue of the SD rats. Enzyme-linked immunosorbent assay was used to evaluate plasma protein levels. Real-time reverse transcription polymerase chain reaction was used to determine messenger RNA (mRNA) expression.
Results: In newborn SD rats, hyperoxia intervention within 7 days may result in acute lung damage. In the plasma and tissue of newborn SD rats, hyperoxia induction may raise levels of HE4, matrix metalloproteinases (MMP) 9 and tissue inhibitors of metalloproteinases (TIMP) 1. We discovered that the HE4 protein activates the phosphorylation of extracellular regulated protein kinases (ERK) and p65, activates the downstream MMP9 signalling pathway, inhibits MMP9 mRNA expression, inhibits protein activity, reduces type I collagen degradation, increases collagen secretion and promotes matrix remodelling and fibrosis in neonatal rat primary alveolar type II epithelial cells by overexpressing and silencing the HE4 gene.
Conclusion: Through the ERK, MMP9 and TIMP1 signalling pathways, HE4 mediates the pathophysiological process of hyperoxia-induced lung damage in rats. Lung damage and lung basal remodelling are mediated by HE4 overexpression.
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