Isolation, Substrate Specificity, and Subunit Characterization of the Xylanosomes Produced by Oerskovia turbata JCM 3160.

This work aims at functional studies of the multienzyme complexes produced by Oerskovia turbata JCM 3160 and reveal of their subunit structures. The multienzyme complexes were isolated, enzymatic assayed, the whole genome sequence was determined in fine scale, and the subunit structure was identified by Maldi-TOF mass spectrometry. The isolated multienzyme complexes here show similar particle size with the xylanosomes produced by Cellulosimicrobium cellulans F16, have at least two conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions. This is the first report of the enzymatic activities and subunit structures of xylanosome produced by Oerskovia turbata, providing insights into its diverse capability as well as degrading bias on hemicelluloses.

Isolation and subunit structure of the xylanosome complex produced by Actinotalea fermentans JCM9966.

Xylanosomes, also known as hemicellulosomes, are hemicellulose-degrading nano-scale multienzyme complexes produced by some Firmicutes, Actinobacteria, and Fungi. Here we report the isolation of the MECs produced by Actinotalea fermentas JCM9966, as well as the functional studies and subunit structure revealed by proteomic identifications. The isolated MECs here shows similar particle size with the xylanosomes produced by C. cellulans F16, have several conserved multi-domain proteins, while differ significantly in enzymatic activities and low molecular weight subunit compositions, indicating diverse capability as well as bias in degrading hemicelluloses.

Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

Evaluation of RT-LAMP Assay for Rapid Detection of SARS-CoV-2.

OBJECTIVE: To evaluate the accuracy of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in community or primary-care settings. METHOD: We systematically searched the Web of Science, Embase, PubMed, and Cochrane Library databases. We conducted quality evaluation using ReviewManager software (version 5.0). We then used MetaDisc software (version 1.4) and Stata software (version 12.0) to build forest plots, along with a Deeks funnel plot and a bivariate boxplot for analysis. RESULT: Overall, the sensitivity, specificity, and diagnostic odds ratio were 0.79, 0.97, and 328.18, respectively. The sensitivity for the subgroup with RNA extraction appeared to be higher, at 0.88 (0.86-0.90), compared to the subgroup without RNA extraction, at 0.50 (0.45-0.55), with no significant difference in specificity. CONCLUSION: RT-LAMP assay exhibited high specificity regarding current SARS-CoV-2 infection. However, its overall sensitivity was relatively moderate. Extracting RNA was found to be beneficial in improving sensitivity.

Relationship between Coronary Artery Tortuosity and Cardiorespiratory Fitness in Patients without Obstructed Coronaries.

OBJECTIVE: This study aimed to analyze the relationship between cardiorespiratory fitness (CRF) and the increasing severity of coronary artery tortuosity (CAT) in patients with non-stenosed coronaries. METHODS: A total of 396 patients who underwent coronary angiography and cardiopulmonary exercise testing (CPET) between August 2020 and July 2021 were included in this single-center retrospective study after excluding patients with significant coronary artery disease (≥50% stenosis). Patients were divided into two groups: no or mild coronary artery tortuosity (N/M-CAT) and moderate to severe coronary artery tortuosity (M/S-CAT) and laboratory electrocardiographic, echocardiographic, and CPET parameters were compared between two groups. RESULTS: M/S-CAT was found in 46.9% of the study participants, with 66.7% being women. M/S-CAT was significantly associated with advanced age (P=0.014) and females (P=0.001). Diastolic dysfunction parameters, E velocity (P=0.011), and E/A ratio (P=0.004) also revealed significant differences between the M/S-CAT group and N/M-CAT group. VO2@peak (1.22±0.39 vs. 1.07±0.39, P<0.01) and VO2@AT (0.77±0.22 vs. 0.71±0.21, P=0.017) were significantly lower in the M/S-CAT group than in the N/M-CAT group. Multivariate logistic regression analysis identified females (OR=0.448; 95% CI, 0.296-0.676; P=0.000) and E/A ratio (OR=0.307; 95% CI, 0.139-0.680; P=0.004) to be independent risk factors of M/S-CAT and showed no association of CPET parameters to M/S-CAT. CONCLUSION: The results indicate that increasing severity of CAT is strongly associated with female gender and E/A ratio and is not directly correlated with decreasing CRF. Further research with a larger patient population and a longer follow-up time is required to fully comprehend the impact of CAT on CRF.

High-density lipoprotein cholesterol and in-hospital mortality in patients with acute aortic dissection.

The association between high-density lipoprotein cholesterol (HDL-C) and mortality in patients with acute aortic dissection (AAD) is unclear. From January 2007 to January 2014, a total of 928 consecutive AAD patients who were admitted within 48 h after the onset of symptoms were enrolled in the study. Patients were divided into two groups according to whether serum HDL-C level was below the normal lower limit or not. The Cox proportional hazard regression model was used to identify the predictive value of HDL-C for in-hospital mortality in patients with AAD. As compared with normal HDL-C group (n=585), low HDL-C group (n=343) had lower levels of systolic blood pressure and hemoglobin and higher levels of leukocyte, alanine aminotransferase, blood glucose, blood urea nitrogen, creatinine and urea acid. Low HDL-C group had significantly higher in-hospital mortality than normal HDL-C group (21.6% vs. 12.6%, log-rank=10.869, P=0.001). After adjustment for baseline variables including demographics and biologic data, the increased risk of in-hospital mortality in low HDL-C group was substantially attenuated and showed no significant difference (adjusted hazard ratio, 1.23; 95% confidence interval, 0.86-1.77; P=0.259). Low HDL-C is strongly but not independently associated with in-hospital mortality in patients with AAD.

CYP2C8-derived epoxyeicosatrienoic acids decrease oxidative stress-induced endothelial apoptosis in development of atherosclerosis: Role of Nrf2 activation.

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 µmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.

Molecular characterization and functional divergence of two Gadd45g homologs in sex determination in half-smooth tongue sole (Cynoglossus semilaevis).

The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) is known to play a major role in embryonic development and sex determination. In this study, two Gadd45g genes were isolated from half-smooth tongue sole (Cynoglossus semilaevis). Using chromosomal fluorescence in situ hybridization (FISH), Gadd45g1 and Gadd45g2 were located on the W and Z chromosomes, respectively. The full-length cDNA sequences of Gadd45g1 (1270bp) and Gadd45g2 (1181bp) were predicted to contain a 480-bp coding sequence that could encode a protein of 159 amino acids residues. A phylogenetic tree showed that the predicted Gadd45g1 and Gadd45g2 amino acid sequences clustered closely in one branch. It is proposed that Gadd45g1 and Gadd45g2 are paralogous genes derived from the divergence of the sex chromosome. Ka/Ks ratios indicated that Gadd45g1 and Gadd45g2 may have undergone a high number of mutations and have a divergence time of only about 68,000years, although Gadd45g homologs are highly conserved. The qRT-PCR demonstrated that Gadd45g1 and Gadd45g2 were highly expressed in ovary, and negligibly expressed in testis of male and neo-male. During development of the ovary (from 80 to 150days), the expression levels of both genes reached high levels. Gadd45g1 was also highly expressed at 50days, the stage just before gonad differentiation in C. semilaevis. All these findings imply functional divergence of the two Gadd45g homologs; Gadd45g1 may be necessary for sex differentiation in the early stage of gonad development, and then Gadd45g1 and Gadd45g2 maintain ovary development and the female character of half-smooth tongue sole.

[Apoptosis in neonatal rat lung exposed to hyperoxia].

OBJECTIVE: Exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia (BPD). Cell death from hyperoxic injury may occur through either an apoptotic or nonapoptotic pathway. The aim of the present study was to investigate the effect of hyperoxia on caspase-3 and p53 gene expression and apoptosis in the lungs of neonatal rats, so as to determine the type of cell death that occurs in the lungs of neonatal rats exposed to hyperoxia. METHODS: Hyperoxic lung injury model was established by exposing to 95% O(2) in the neonatal period of Spraque-Dawley rats. The levels of caspase-3 mRNA and p53 mRNA expression in the lungs of neonatal rats exposed to 95% hyperoxia or room air were detected by RT-PCR. To quantify PCR products, PCR products were electrophoretically separated with 1.5% agarose gels. The optical density (A) values of the DNA bands were quantified by complete gel documentation and analysis system. The A ratios of p53/beta-actin denoted the relative content of p53 mRNA, results were showed as mean +/- standard deviation. The specific positive or negative bands of caspase-3 in electrophoresis gels were counted, Fisher's exact test of propabilities was used to determined statistically significant differences between two groups. We determined the extent of apoptosis taking place in the lungs of neonatal rats exposed to 95% hyperoxia using terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate-fluorescence nick-end labeling (TUNEL) in 7-d-old neonatal lung. Under light microscope, five areas of lung parenchyma were systematically and randomly photographed from each animal and positive cells among 500 lung cells were calculated. Results were showed as mean +/- standard deviation. RESULTS: We found increased levels of p53 messenger RNA, a gene associated with apoptosis, in the lungs of neonatal rat born and raised in 95% hyperoxia. Moderate increase in the level of p53 mRNA was found in the hyperoxic-treated group at 24 h (q = 3.2305, P > 0.05). Significant increase in the level of p53 mRNA was found in the hyperoxic-treated group at 48 h (q = 7.2941, P < 0.01). The levels of p53 mRNA expression in neonatal rat lungs exposed to 95% O(2) for 72 h or 96 h returned to normal level. The levels of caspase-3 mRNA expression were very low or absent in the hyperoxic-treated groups at 12 h, 24 h, 48 h, 72 h and 96 h or in the air-breathing groups at 12 h, 24 h, 48 h, 72 h and 96 h. An increase in the number of cells undergoing apoptosis was found in the hyperoxic-treated group at 7 d (F = 56.5010, P < 0.001) which was significantly greater than the number of apoptotic cells found in the lungs of rats of the same age exposed to room air. CONCLUSION: Our results suggested that 95% hyperoxia could temporarily up-regulate the gene expression of p53, which induced the transcription of p21(WAF/CIP1) mRNA. Furthermore, p21(WAF/CIP1) could lead to cell cycle arrest and inhibit proliferation of lung cells. Meanwhile, p53 could also promote apoptosis of lung cells. Therefore, exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia (BPD), and hyperoxia may affect the future lung growth and lead to barrier of lung development. The treatments of anti-apoptosis and promoting alveoli growth hold a promising perspective in hyperoxic lung injury. The level and ratio of caspase-3 gene expression were very low or absent in the lungs of neonatal rats exposed to 95% O(2) or room air. We speculated that caspase-3 gene expression was not essential in the hyperoxia induced lung cell apoptosis in neonatal rats.

[Effects of U74389G on pulmonary macrophage influx and lung development in 95% O2 exposed newborn rats].

OBJECTIVE: Oxygen toxicity is believed to play a critical role in the pathogenesis of bronchopulmonary dysplasia (BPD). U74389G, a potent 21-aminosteroid antioxidant, was applied to the 95% O(2) induced acute lung injury in newborn rat model. The present study aimed to investigate the mechanism of hyperoxic lung injury and the interaction of possible mediators, and to explore the effect of antioxidant intervention. METHODS: Newborn Sprague-Dawley rats were randomly divided into four groups: air-exposed control, air-exposed treated with U74389G, hyperoxia-exposed control, hyperoxia-exposed treated with U74389G. Hydroxyl radical formation (2,3-DHBA and 2,5-DHBA) was assessed by an aromatic hydroxylation assay using GC/MS with salicylate as the probe. The 8-isoprostane, a specific marker for in vivo lipid peroxidation, was quantitated by enzyme immunoassay. Pulmonary macrophage influx and nitrotyrosine formation were measured by means of immunohistochemistry. (3)H-TdR (autoradiography) incorporation was assessed as an index of active lung cell growth. RESULTS: Exposure to 95% O(2) for 7 days induced significant lung injury and mortality. The contents of hydroxyl radical in the hyperoxia-exposed lungs were dramatically increased [(2,3-DHBA 49.2 +/- 3.5 pmol/mg), (2,5-DHBA 55.8 +/- 2.3 pmol/mg), P < 0.05) and were decreased by treatment with U74389G [(2,3-DHBA 37.9 +/- 2.4 pmol/mg), (2,5-DHBA 31.3 +/- 1.9 pmol/mg), P < 0.05). The level of 8-isoprostane in the lungs of 95% O(2)-exposed newborn rats was significantly raised (546.6 +/- 32.2 pg/mg, P < 0.05) and lowered down by U74389G (358.5 +/- 24.1 pg/mg, P < 0.05). This phenomenon was also observed in the air-exposed animals. Remarkable pulmonary macrophage infiltration was evident in hyperoxia-exposed newborn rats and was attenuated by U74389G treatment. Nitrotyrosine distributed in the lung parenchyma and epithelial cells of large airway of hyperoxia-exposed newborn rats. The extent of protein nitration was reduced by U74389G, but the oxygen induced morphological change was not significantly improved by U74389G treatment. Exposure to 95% O(2) induced lung growth arrest as shown by (3)H-TdR incorporation. U74389G partially preserved active lung cell growth in hyperoxia-exposed rats, but showed an inhibitory effect on normal lung cell growth. CONCLUSION: Through scavenging hydroxyl radical and lipid peroxides, U74389G could block pulmonary macrophage influx and partly avert alveolar development arrest in hyperoxia-exposed newborn rats. Antioxidant intervention holds promising in hyperoxic lung injury though cautions should be taken as possible interference on normal cell development.

[Application of gas chromatography-mass spectrometry analysis on urine filter paper in the high-risk screening and diagnosis of inherited metabolic diseases].

OBJECTIVE: To establish a specific procedure for the high-risk screening and diagnosis of organic acidurias and other inherited metabolic diseases in China. METHODS: A nation-wide network for the high-risk screening and diagnosis of genetic metabolic diseases was formed to facilitate the collaboration. Urine samples were collected using filter paper from patients with clinical symptoms suspicious of inherited metabolic diseases. The samples were eluted with distilled water and internal standards were added. Samples were treated with hydroxylamine hydrochloride to form oximes to improve the recoveries of 2-ketoacids. Urinary organic acids were extracted with ethyl acetate and diethyl ether under acidic condition. After dehydration, the combined organic phase was evaporated to dryness with nitrogen. The residues were added with BSTFA + 1%TMCS and heat incubated to form the trimethylsilyl derivatives, and then were analyzed on an Agilent 5890/5973N gas chromatography-mass spectrometer (GC-MS), with a 7683 series auto-sampler. The peaks were identified by reference to a mass spectral library. RESULTS: Totally 352 samples were collected from the network collaborating hospitals since 2001. Thirty-four (9.66%) cases of various inherited metabolic diseases were diagnosed with an age range of 2 days to 14 years. The disease profile was consisted of methylmalonic acidemias (6), alpha-keto-glutaric aciduria (5), tyrosinemia type I (4), dicarboxylic aciduria (4), multiple carboxylase deficiency (3), phenylketonuria (3), lactic acidemia (3), propionic acidemia (2), ornithine transcarbamoylase deficiency (1), ethylmalonic-adipic aciduria (1), glutaric aciduria type II (1) and 3-methylcrotyl CoA carboxylase deficiency (1). The most common clinical symptoms and signs included mental and developmental retardation, convulsion, musculotonic abnormality and jaundice. Routine laboratory tests often revealed metabolic acidosis, hypoglycemia and hyperammonemia, etc. CONCLUSION: Urine organic acids analysis by GC-MS remains to be the most important technique for the high-risk screening and diagnosis of inherited metabolic diseases. Use of urine filter paper for sample collection and analysis in advanced genetic metabolic centers is a practical approach to extend the diagnostic capacity and improve the management of such diseases in China. Collaborative network played a critical role in the success of the program.