Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

An overview of current advances in perinatal alcohol exposure and pathogenesis of fetal alcohol spectrum disorders.

The adverse use of alcohol is a serious global public health problem. Maternal alcohol consumption during pregnancy usually causes prenatal alcohol exposure (PAE) in the developing fetus, leading to a spectrum of disorders known as fetal alcohol spectrum disorders (FASD) and even fetal alcohol syndrome (FAS) throughout the lifelong sufferers. The prevalence of FASD is approximately 7.7 per 1,000 worldwide, and is even higher in developed regions. Generally, Ethanol in alcoholic beverages can impair embryonic neurological development through multiple pathways leading to FASD. Among them, the leading mechanism of FASDs is attributed to ethanol-induced neuroinflammatory damage to the central nervous system (CNS). Although the underlying molecular mechanisms remain unclear, the remaining multiple pathological mechanisms is likely due to the neurotoxic damage of ethanol and the resultant neuronal loss. Regardless of the molecular pathway, the ultimate outcome of the developing CNS exposed to ethanol is almost always the destruction and apoptosis of neurons, which leads to the reduction of neurons and further the development of FASD. In this review, we systematically summarize the current research progress on the pathogenesis of FASD, which hopefully provides new insights into differential early diagnosis, treatment and prevention for patents with FASD.

miR-317-3p and miR-283-5p Play a Crucial Role in Regulating the Resistance to Indoxacarb in Spodoptera frugiperda by Targeting GSTs4.

Spodoptera frugiperda is primarily controlled through chemical insecticides. Our RNA-seq data highlight the overexpression of GSTs4 in indoxacarb-resistant S. frugiperda. However, the exact role of GSTs4 in indoxacarb resistance and its regulatory mechanisms remains elusive. Therefore, we investigated the functional role of GSTs4 in S. frugiperda and explored the underlying post-transcriptional regulatory mechanisms. GSTs4 was highly overexpressed (27.6-fold) in the indoxacarb-resistant strain, and GSTs4 silencing significantly increases the susceptibility of S. frugiperda to indoxacarb, increasing mortality by 27.3%. miR-317-3p and miR-283-5p can bind to the 3'UTR of GSTs4, and the targeting relationship was confirmed by dual-luciferase reporter assays. Injecting miR-317-3p and miR-283-5p agomirs reduces GSTs4 levels by 64.8 and 42.3%, respectively, resulting in an increased susceptibility of S. frugiperda to indoxacarb. Conversely, the administration of miR-317-3p and miR-283-5pantagomirs increases GSTs4 expression and reduces larval susceptibility to indoxacarb. These findings demonstrate that miR-317-3p and miR-283-5p contribute to indoxacarb resistance in S. frugiperda by regulating the overexpression of GSTs4.

Treatment of Pulp Canal Obliteration Using a Dynamic Navigation System: Two Case Reports.

Endodontic treatment of calcified canals presents a major challenge because of the high incidence of complications, such as perforation, canal geometry alteration, and loss of dental hard tissue. The dynamic navigation technique uses an optical tracking system for real-time navigation to guide the operator to drill according to the preoperative plan and obtain access to the calcified canals. This article describes in detail the use, advantages, disadvantages, and limitations of a novel dynamic navigation system in 2 cases with severely calcified canals. The findings in these cases demonstrate that dynamic navigation system is a promising technique for locating calcified root canals.

EIF4E over-expresses and enhances cell proliferation and cell cycle progression in nasopharyngeal carcinoma.

Eukaryotic translation initiation factor 4E (eIF4E) is involved in integration and amplification of many carcinogenesis signals in tumors. However, it remains unclear whether eIF4E over-expresses in NPC and whether it is associated with the development of NPC. Here, we analyzed the expression state of eIF4E, c-Myc, and MMP9 in 24 nasopharyngitises and 64 nasopharyngeal carcinomas (NPC) tissues and studied the influences of eIF4E on the proliferation and cell cycle in NPC cell lines. The results indicate that eIF4E might over-express in NPC and the over-expression of eIF4E promotes NPC growth and cell cycle progression through enhancing the translational expression of c-Myc and MMP9. The finding certainly adds new knowledge in the understanding of the carcinogenesis of NPC and provides a potential molecular target for the NPC therapy and prevention.

Issues pertaining to the analysis of buprenorphine and its metabolites by gas chromatography-mass spectrometry.

"Substitution therapy" and the use of buprenorphine (B) as an agent for treating heroin addiction continue to gain acceptance and have recently been implemented in Taiwan. Mature and widely utilized gas chromatography-mass spectrometry (GC-MS) technology can complement the low cost and highly sensitive immunoassay (IA) approach to facilitate the implementation of analytical tasks supporting compliance monitoring and pharmacokinetic/pharmacogenetic studies. Issues critical to GC-MS analysis of B and norbuprenorphine (NB) (free and as glucuronides), including extraction, hydrolysis, derivatization, and quantitation approaches were studied, followed by comparing the resulting data against those derived from IA and two types of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Commercial solid-phase extraction devices, highly effective for recovering all metabolites, may not be suitable for the analysis of free B and NB; acetyl-derivatization products exhibit the most favorable chromatographic, ion intensity, and cross-contribution characteristics for GC-MS analysis. Evaluation of IA, GC-MS, and LC-MS/MS data obtained in three laboratories has proven the 2-aliquot GC-MS protocol effective for the determination of free B and NB and their glucuronides.

An empirical study on the selection of analytes and corresponding cutoffs for immunoassay and GC-MS in a two-step test strategy--buprenorphine example.

(i) Standard solutions of buprenorphine (B) and three metabolites; (ii) immunoassay (IA) reagents designed for the analysis of B and/or its metabolites; and (iii) clinical urine specimens collected from patients (under B-treatment), constitute the B-System for fundamental study of parameters critical to the two-step test strategy, an analytical approach designed for a high-volume testing environment. The cross-reacting characteristics of IA reagents were examined using standard solutions of B and its metabolites. Resulting data were used as the basis for selecting target analytes suitable for the preliminary and the confirmatory test steps. Test data derived from IA and GC-MS analysis of clinical urine specimens (with natural distribution of B and its metabolites) were quantitatively correlated. Correlation parameters were examined: (i) to verify whether the analyte-pair targeted by the IA and GC-MS test steps has been properly selected; and (ii) to decide on appropriate cutoffs for the two test steps. In conclusion, this study has demonstrated that the most effective analyte(s) that should be targeted in the GC-MS determination step vary with the IA selected in the preliminary test step. All analytes that generate significant responses to the IA reagent should be targeted in the GC-MS test step.

A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography-mass spectrometry.

A two-step derivatization approach has been developed to enable the simultaneous analysis of glyoxal, methylglyoxal, and 3-deoxyglucosone by the most efficient and widely applied GC-MS methodology. These three analytes are reactive carbonyl compounds associated with the formation of advanced glycation and lipoxidation end products, a process thought to contribute to uremic toxicity and referred to as "carbonyl stress". Effective analysis of these compounds would facilitate understanding these compounds' role in diabetes-related complications. Plasma samples were deproteinized by acetonitrile, followed by a two-step derivatization approach. Pooled plasma samples from healthy individuals were used as the "blank" for preparing calibration standards. The concentrations of the analytes in the "blank" were first determined by standard addition method. Calibration parameters were accordingly established and used to analyze these compounds in plasma samples collected from healthy individuals and diabetic patients. Analytical findings are comparable with those reported in the literature. Quantitation data can be further improved by making available and using isotopically labeled analogs of these analytes as the internal standards.