The Influences of Cryopreservation Methods on RNA, Protein, Microstructure and Cell Viability of Skeletal Muscle Tissue.

Background: Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. Methods: We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN2) snap-freezing, LN2-cooled isopentane snap-freezing, RNAlater®-based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. Results: PCSF preserved RNA integrity better than LN2 and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN2. The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. Conclusion: PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.

Variable Control and Its Influence Before Urine Sample Analysis in a Field Environment.

Background and Objectives: The aim of the study was to store urine samples at different temperatures and humidity levels and analyze common biochemical test results and point-of-care testing (POCT) indicators according to different storage times and evaluate whether the samples should be centrifuged to study the best storage conditions for urine samples. Methods: Random midstream urine samples (100 mL) were collected from 10 healthy individuals. A portion of the samples was centrifuged. The remaining samples were not centrifuged and were stored under different temperature and humidity conditions for different periods. We measured urine indicators ([Na+], [K+], [Cl-], gamma-glutamyl transpeptidase [GGT], urea, and creatinine [Cr]) at 2, 4, 24, and 72 hours and 7 and 55 days, and we used POCT to measure myoglobin (Mb) and microalbumin (mAlb) concentrations. Results: Centrifugation of urine samples decreased the measured GGT and increased the measured Mb. In urine samples stored at 4°C and room temperature, electrolyte concentrations were scarcely affected by storage time. After storage at 50°C for 24 hours, the measured [Na+] and [Cl-] levels changed. Metabolites (urea and Cr) underwent no obvious change across temperatures. GGT did not change during long-term storage at 4°C. The mAlb level changed significantly only after storage at 4°C. When stored at 4°C, Mb changed little within 4 hours. Under humid conditions, [Na+] and [Cl-] increased significantly after 24 hours, and urea decreased significantly after 7 days of storage. Under dry storage conditions, urinary Cr and GGT decreased, and under humid conditions, these concentrations increased. At high humidity, mAlb increased significantly after 72 hours. Conclusions: Electrolyte and amino acid metabolite concentrations were less affected by storage time at 4°C and room temperature than at other temperatures. Some proteins are sensitive to environmental changes; samples collected for quantification of these proteins can be stored briefly at 4°C after centrifugation. Normal humidity conditions meet most physiological testing requirements.

Estrogen ameliorates sepsis-induced vascular hyporeactivity in thoracic aorta of female rats via permissive effect of GRα expression.

OBJECTIVE: Estrogen is correlated to the lower mortality and disease severity of female than that of male, which indicates the potential therapeutic role of estrogen supplement therapy in sepsis. The structure of Daidzein is similar to that of 17ß estradiol (E2), an estrogen in human body, causing the exogenous Daidzein can interact with estrogen receptor as well as E2 in the body. We aim to explore the therapeutic role of estrogen in sepsis-induced vascular dysfunction. Also, we wonder if estrogen regulates blood pressure via glucocorticoid-mediated vascular reactivity. METHODS: Female SD rats received ovariectomy (OVX) to induce estrogen deficiency. After 12 weeks of administration, cecal ligation and puncture (CLP) was used to establish the in vivo model of sepsis. Lipopolysaccharide (LPS) was used to construct the in vitro model of sepsis in vascular smooth muscle cells (VSMCs). E2 and Daidzein were used for estrogen supplement therapy. RESULTS: E2 and Daidzein significantly inhibited inflammation infiltration and histopathological injury in thoracic aorta in the rat model with CLP. E2 and Daidzein improved carotid pressure and vascular hyporeactivity in sepsis rats with OVX. Importantly, E2 and Daidzein promoted glucocorticoid permissive action and increased glucocorticoid receptor α (GRα) expression in thoracic aorta smooth muscle cells. E2 and Daidzein upregulated GRα, and inhibits cytokine production, proliferative phenotype and cell migration in LPS-induced VSMCs. CONCLUSION: Estrogen improved vascular hyporeactivity in thoracic aorta induced by sepsis via permissive effect of GRα expression.

Integrated Analysis of the Alterations in Gut Microbiota and Metabolites of Mice Induced After Long-Term Intervention With Different Antibiotics.

Objectives: We aimed to study the effect of antibiotic-induced disruption of gut microbiome on host metabolomes and inflammatory responses after long-term use of antibiotics. Methods: A total of three groups of 3-week-old female C57BL/6 mice (n = 44) were continuously treated with vancomycin (VAN), polymyxin B (PMB), or water, respectively, for up to 28 weeks. Fecal samples collected at different time points were analyzed by bacterial 16S rRNA gene sequencing and untargeted metabolomics by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS). Serum cytokines (IFN-γ, IL-2, IL-10, IL-13, IL-17A, and TNF-α) were determined by multiplex immunoassay. Results: Treatment by VAN or PMB did not affect the average body weight of mice. However, a heavier caecum observed in VAN-treated mice. Compared with PMB-treated and control mice, VAN treatment induced more rapid dysbiosis of gut microbiota and dysmetabolism. Instead of Bacteroides, VAN-treated mice had a compositional shift to Proteobacteria and its species Escherichia coli and Verrucomicrobia and its species Akkermansia muciniphila. The shift was accompanied by decreased richness and diversity in microbiota. PMB-treated mice had an increased Firmicutes, and the diversity was shortly increased and further decreased to the baseline. Decreased levels of short-chain and long-chain fatty acids, bile acids, L-arginine, dopamine, L-tyrosine, and phosphatidylcholine (all p < 0.05) were observed in VAN-treated mice. In contrast, significantly increased levels of amino acids including L-aspartic acid, beta-alanine, 5-hydroxy-L-tryptophan, L-glutamic acid, and lysophosphatidylcholines (all p < 0.05) were found. These changes occurred after 3-week treatment and remained unchanged up to 28 weeks. For PMB-treated mice, metabolites involved in the metabolic pathway of vitamin B6 were decreased, whereas glycocholic acid and chenodeoxycholic acid were increased (all p < 0.05). After 8-week treatment, VAN-treated mice had significantly higher levels of serum IFN-γ, IL-13, and IL-17A, and PMB-treated mice had higher levels of IL-13 and IL-17 compared to control mice. At 28-week treatment, only IL-17A remained high in PMB-treated mice. Conclusion: This study showed that the antibiotic-induced alterations in gut microbiota contribute to host inflammatory responses through the change in metabolic status, which are likely related to the type, rather than timing of antibiotic used.

Identification of novel variations in SLC6A8 and GAMT genes causing cerebral creatine deficiency syndrome.

Cerebral creatine deficiency syndromes (CCDSs) are a group of rare mendelian disorders mainly characterized by intellectual disability, movement anomaly, behavior disorder and seizures. SLC6A8, GAMT, and GATM are known genes responsible for CCDS. In this study, seven pediatric patients with developmental delay were recruited and submitted to a series of clinical evaluation, laboratory testing, and genetic analysis. The clinical manifestations and core biochemical indications of each child were basically consistent with the diagnosis of CCDS. Genetic diagnosis determined that all patients were positive for SLC6A8 or GAMT variation. A total of 12 variants were identified in this cohort, including six novel ones. The frequency of these variants, the Revel scores and the conservatism of the affected amino acids support their pathogenicity. Our findings expanded the mutation spectrum of CCDS disorders, and provided solid evidence for the counseling to affected families.

Identification of the Signature Associated With m6A RNA Methylation Regulators and m6A-Related Genes and Construction of the Risk Score for Prognostication in Early-Stage Lung Adenocarcinoma.

BACKGROUND: N6-methyladenosine (m6A) RNA modification is vital for cancers because methylation can alter gene expression and even affect some functional modification. Our study aimed to analyze m6A RNA methylation regulators and m6A-related genes to understand the prognosis of early lung adenocarcinoma. METHODS: The relevant datasets were utilized to analyze 21 m6A RNA methylation regulators and 5,486 m6A-related genes in m6Avar. Univariate Cox regression analysis, random survival forest analysis, Kaplan-Meier analysis, Chi-square analysis, and multivariate cox analysis were carried out on the datasets, and a risk prognostic model based on three feature genes was constructed. RESULTS: Respectively, we treated GSE31210 (n = 226) as the training set, GSE50081 (n = 128) and TCGA data (n = 400) as the test set. By performing univariable cox regression analysis and random survival forest algorithm in the training group, 218 genes were significant and three prognosis-related genes (ZCRB1, ADH1C, and YTHDC2) were screened out, which could divide LUAD patients into low and high-risk group (P < 0.0001). The predictive efficacy of the model was confirmed in the test group GSE50081 (P = 0.0018) and the TCGA datasets (P = 0.014). Multivariable cox manifested that the three-gene signature was an independent risk factor in LUAD. Furthermore, genes in the signature were also externally validated using the online database. Moreover, YTHDC2 was the important gene in the risk score model and played a vital role in readers of m6A methylation. CONCLUSION: The findings of this study suggested that associated with m6A RNA methylation regulators and m6A-related genes, the three-gene signature was a reliable prognostic indicator for LUAD patients, indicating a clinical application prospect to serve as a potential therapeutic target.