Experimental Biology and Medicine

The application of a bronchoconstrictor, usually Methacholine (MCh), in respiratory mechanics studies is usually accompanied by the assessment of respiratory mechanics in a dose–response curve. The MCh used in the dose–response curve can be inhaled (i.h.) and intravenous (i.v.) and there are studies comparing i.v. bolus and i.h. MCh in both mice and rats. However, MCh i.v. can be injected at short time interval (bolus) or in continuous infusion. This comparison is relevant since the way MCh is applied influences the mathematical model. We chose an aging process scenario to compare both protocols. This study aims to compare respiratory mechanics of 3-, 6-, and 10-month SAMR1 mice and how both administration methods (continuous infusion and bolus) impact respiratory mechanics evaluation. Both protocols were capable of assessing the difference among ages and doses in: peak or plateau; and area under the curve analysis. The respiratory mechanics parameters were Rn, G, and H (two-way analysis of variance: groups and doses with a P < 0.05 for all). Also, the infusion protocol presented a higher sensitivity to dose increment. In conclusion, both protocols were able to discriminate intragroup and intergroup differences. In the bolus protocol, the highest value of each curve dose may not correspond to the highest real value, and the loss of this point may be a problematic factor in the sample size. These factors are not present in the infusion protocol. Additionally, at this lineage and age screening, the infusion protocol appeared to be more sensitive to differences among ages when compared to the bolus protocol.

Chinese hamster ovary cells are the predominant cell lines used for bio-therapeutic production. Real-time quantitative PCR (RT-qPCR) and transcriptomics are powerful tools to understand and optimize the Chinese hamster ovary cells for higher productivity or better control of product qualities. Reliable reference genes, which were proved to be experiment-specific, are critical yardsticks. In this study, we compared expression stability of 20 candidate reference genes at mRNA level, including commonly used housekeeping genes, previous literature reported genes in Chinese hamster ovary cells producing an intact antibody, and new candidates suggested by our RNA-seq transcriptomic database, in RT-qPCR reactions in Fc-fusion protein-producing Chinese hamster ovary cells with various productivity during long-term cultivation and fed-batch cultures at 26 different conditions. geNorm, NormFinder, BestKeeper, and ΔCt programs and methods were utilized to analyze the gene expression stability and gave an overall ranking. Akr1a1, Gpx1, and Aprt in long-term cultivation and Akr1a1, Rps16 in fed-batch culture, which have not been reported previously, exhibited the highest stability of gene expression, while Pabpn1, Hirip3, and Actb in both sets of experiments together with Atp5f1 in long-term passage process showed the weakest stability. The results were then validated using GLP1-Fc (Glucagon-like peptide-1 Fc fusion protein) gene as the target with determined expression level which were doubly confirmed by both absolute RT-qPCR and confocal microscopy. These new references should be considered for the investigations on Chinese hamster ovary cells in related research.

The occurrence and acuteness of liver cirrhosis were strongly associated with the hepatocarcinogenesis and the prognosis of hepatocellular carcinoma (HCC). This study compared the prognostic significance of non-invasive fibrosis panel containing 15 indices in hepatitis-B-associated HCC patients’ post-curative resection. Four hundred and five consecutive hepatitis-B-related HCC patients who went through curative hepatectomy were investigated retrospectively. The multivariate Cox proportional hazard model was used to evaluate independent prognostic factors for overall survival (OS). The accuracy in diagnosis of each non-invasive fibrosis index in cirrhosis detection was determined by the area under receiver operating characteristic (AUC) curve. Preoperative AST to platelet ratio index (APRI), Goteburg University Cirrhosis Index (GUCI), and King Score all exhibited a superior performance in diagnosis of cirrhosis detection with AUC > 0.7. APRI and Fibro-α Score were the risk factors that behaved independently in predicting the OS of HCC patients, with an hazard ratio (HR) value of 1.550 (P = 0.012) and 1.420 (P = 0.033), respectively. Preoperative APRI was a relatively accurate predictor of cirrhotic status and prognosis in HCC due to hepatitis-B among the 15 non-invasive fibrosis indices.

This study investigated ovarian expressions of bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), and C-KIT according to age in female mice to determine whether these factors can be served as new potential biomarkers of ovarian aging. Ovaries were collected from mice aged 10, 20, 30, and 40 weeks, and ovarian expressions of BMP15, GDF9, and C-KIT were examined by real-time PCR, Western blot, and immunohistochemistry. Follicle counts were measured on histological hematoxylin and eosin staining. In the second experiment to evaluate ovarian function, after superovulation with PMSG and hCG, the numbers of zygotes retrieved and embryo development rate were examined. Ovarian expressions of BMP15, GDF9, and C-KIT decreased with age. Follicle counts, numbers of retrieved zygotes, and embryo development rate were also significantly reduced in old mice over 30 weeks compared with young mice. These results indicate that these factors could be served as new potential biomarkers of ovarian aging.

Gene mutations are closely related to cancers and drug sensitivity. Noninvasive liquid biopsy was used to detect mutations of ctDNA in plasma, which is regarded as an indicator of chemotherapy reaction. In this study, we performed exon sequencing of 416 cancer-related genes for cancer primary tissue and plasma samples of 20 patients in 11 cancers. The comprehensive mutation landscape was obtained by bioinformatics tools. In all samples, a total of 0–135 genes involved somatic mutations, and 5–209 genes involved copy number variation. APC, KRAS, and TP53 were detected as frequently mutated genes. Nineteen genes with high-frequency copy-number amplification and 59 with frequent copy-number deletions were identified. By quantitatively assessing the degree of agreement, we found that liquid biopsy is reliable instead of tissues. Besides, 31 mutation prognostic markers in 7 cancers were screened by integrating the consistent mutations and enlarging samples in TCGA. Moreover, from drug-mutation network, 25 drugs connected with 9 mutations (B-Mut-9) were obtained which can be served as drug biomarkers in blood. This was proved by further integrating the mutation information of patients in TCGA into drug-mutation network. In summary, the variation in ctDNA can be used as the biomarkers for cancer prognosis and drug efficacy prediction.

Nicotine dependence is an addiction to tobacco products and a global public health concern that in part would be influenced by our genetics. Smokers are reported to have reduced MAOA activity, but the results from genetic associations with this gene have been inconclusive. Two functionally relevant variable number tandem repeat (VNTR) domains, termed uVNTR and dVNTR, in the MAOA gene are well characterized transcriptional regulatory elements. In the present study, we analyzed uVNTR and dVNTR polymorphisms in the MAOA gene in the Vietnamese male population of smokers and non-smokers in order to assess the association of MAOA with the nicotine dependence measured by the Fagerström Test for Nicotine Dependence (FTND). Individual analysis of VNTRs separately identified uVNTR to be associated with the F6 question of the FTND indicating the stronger addiction to nicotine. No associations were found between the dVNTR and smoking behavior. The combination of dVNTR and uVNTR, that predicts low expression of MAOA (10–3 haplotypes), was significantly associated with the higher nicotine dependence (FTND score), longer smoking duration, and more persistent smoking behavior (fewer quit attempts). In conclusion, our study confirms that low MAOA expression is genetically predictive to the higher nicotine dependence.

The microglia are the resident immune cells in the central nerve system. In the various pathological conditions, prolonged activated microglia could deteriorate brain damage. The regulation of the microglia polarization should be considered in developing an intervention for ischemic stroke patients. Normobaric intermittent hypoxic training protects the brain from intensive ischemic stresses. This study examined the role of intermittent hypoxic training in the regulation of microglia polarization that occurs in the in vitro model of oxygen–glucose deprivation (OGD)–reoxygenation. EOC20 were assigned to the following groups; (1) Normoxia, (2) oxygen–glucose deprivation–reoxygenation, (3) intermittent hypoxic training, (4) oxygen–glucose deprivation–reoxygenation +  intermittent hypoxic training; 24 h after the intermittent hypoxic training, microglia were harvested to perform the following experiments; cell viability (Calcein AM and LDH activity assay), quantification of proteins (Western blot), cytokine (ELISA), and reactive oxygen species (ROS) (H₂DCFDA assays), phagocytic activity by using latex beads coated with FITC, and cell phenotype (immunocytochemistry and flow cytometric analysis, and immunoblot CD206 (M2)). One-way ANOVA with Tukey’s post hoc test was used for the statistical analysis. Oxygen–glucose deprivation/reoxygenation decreases cell viability to 50% of normoxia. Intermittent hypoxic training protects the microglia from oxygen–glucose deprivation/reoxygenation stress. Intermittent hypoxic training regulates the polarization of the microglial phenotype toward anti-inflammatory type M2 (vs. oxygen–glucose deprivation and reoxygenation). Intermittent hypoxic training increases phagocytic activity (about 12 folds) vs. normoxia. ROS in the oxygen–glucose deprivation/reoxygenation group is increased, but intermittent hypoxic training lowers the ROS generation by oxygen–glucose deprivation/reoxygenation. The protein content of the toll-like receptor (TLR2) was significantly elevated in the oxygen–glucose deprivation and reoxygenation group, and intermittent hypoxic training lowered to normoxia level. Anti-inflammatory cytokines, such as IL-10 and IL-4, were significantly increased in the intermittent hypoxic training groups. Due to the effect of intermittent hypoxic training on the microglia phenotype, intermittent hypoxic training could be considered as an effective intervention in the treatment or rehabilitation program for the ischemic stroke victims.

We previously reported on the development of left ventricular (LV) structural and functional changes in an aged, female rat model where the effects of ovariectomy and excess weight (stimulated by fructose in water) were also explored. Ovariectomy and/or excess weight led to a prolongation of active relaxation, loss of cardiac output, and LV fibrosis in the setting of preserved ejection fraction. In this follow-up study, we wished to characterize the possible role of LV inflammation, oxidative stress (OS), and cell death in inducing such changes. Four experimental groups were studied: young (3 months old), aged (18 months old), aged + ovariectomy (OVX), and aged + ovariectomy + 10% fructose (OVF). Using conventional histology and immunohistochemistry of myocardium as well as biochemical assays of plasma samples, we document the presence of inflammatory cell aggregates in LV myocardium which are associated to high levels of plasma inflammatory cytokines (IL-1β, TNF-α, IFN-γ, TGF-β1) and OS (carbonyl proteins) in aged, OVX, and OVF vs. young animals. In the inflammatory areas, normal cardiac tissue was substituted by replacement and interstitial fibrosis and M1 macrophages, (as per by CD68 immunostaining) as we all as by co-localization with TGF-β1. We also document increases in plasma troponin I levels, loss of capillary density, cardiomyocyte hypertrophy, and death. Select changes were further aggravated by ovariectomy and/or excess weight. In conclusion, aging in the female rat heart, when compounded with estrogen depletion and excess weight promotes the development of greater levels inflammation, OS, fibrosis, capillary rarefaction, cardiomyocyte hypertrophy, and injury/death. These factors likely play an important role in the development of LV remodeling that leads to the development of a “pre-HFpEF” phenotype.