Simulated microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts can be prevented by protection of primary cilia.

Oxidative stress has been considered to be closely related to spaceflight-induced bone loss; however, mechanism is elusive and there are no effective countermeasures. Using cultured rat calvarial osteoblasts exposed to microgravity simulated by a random positioning machine, this study addressed the hypotheses that microgravity-induced shortening of primary cilia leads to oxidative stress and that primary cilium protection prevents oxidative stress and osteogenesis loss. Microgravity was found to induce oxidative stress (as represented by increased levels of reactive oxygen species (ROS) and malondialdehyde production, and decreased activities of antioxidant enzymes), which was perfectly replicated in osteoblasts growing in NG with abrogated primary cilia (created by transfection of an interfering RNA), suggesting the possibility that shortening of primary cilia leads to oxidative stress. Oxidative stress was accompanied by mitochondrial dysfunction (represented by increased mitochondrial ROS and decreased mitochondrial membrane potential) and intracellular Ca2+ overload, and the latter was found to be caused by increased activity of Ca2+ channel transient receptor potential vanilloid 4 (TRPV4), as also evidenced by TRPV4 agonist GSK1016790A-elicited Ca2+ influx. Supplementation of HC-067047, a specific antagonist of TRPV4, attenuated microgravity-induced mitochondrial dysfunction, oxidative stress, and osteogenesis loss. Although TRPV4 was found localized in primary cilia and expressed at low levels in NG, microgravity-induced shortening of primary cilia led to increased TRPV4 levels and Ca2+ influx. When primary cilia were protected by miR-129-3p overexpression or supplementation with a natural flavonoid moslosooflavone, microgravity-induced increased TRPV4 expression, mitochondrial dysfunction, oxidative stress, and osteogenesis loss were all prevented. Our data revealed a new mechanism that primary cilia function as a controller for TRPV4 expression. Microgravity-induced injury on primary cilia leads to increased expression and overactive channel of TRPV4, causing intracellular Ca2+ overload and oxidative stress, and primary cilium protection could be an effective countermeasure against microgravity-induced oxidative stress and loss of osteogenic potential of osteoblasts.

Development and validation of a simultaneous method for the analysis of benzothiazoles and organic ultraviolet filters in various environmental matrices by GC-MS/MS.

The presence of benzothiazoles (BTHs) and organic ultraviolet filters (UV filters) in aquatic ecosystems has emerged as a significant environmental issue, requiring urgent and efficient determination methods. A new, rapid, and sensitive determination method using gas chromatography triple quadrupole mass spectrometer (GC-MS/MS) was developed for the simultaneous extraction and analysis of 10 commonly used BTHs and 10 organic UV filters in surface water, wastewater, sediment, and sludge. For aqueous samples, solid-phase extraction (SPE) method was employed with optimizing of SPE cartridge type, pH, and elution solvent. For solid samples, ultrasonic extraction-solid-phase extraction purification (UE-SPE) and pressurized liquid extraction (PLE) methods were compared. And extraction conditions for ultrasonic extraction method (extraction solvents and extraction times) and PLE method (extraction temperatures and extraction cycles) were optimized. The limits of quantification for the 20 target compounds in surface water and wastewater were 0.01-2.12 ng/L and 0.05-6.14 ng/L, while those for sediment and sludge with UE-SPE method were 0.04-5.88 ng/g and 0.22-6.61 ng/g, respectively. Among the 20 target compounds, the recoveries ranged from 70 to 130% were obtained for 16, 15, 15, and 15 analytes in the matrix-spiked samples of surface water, wastewater, sediment, and sludge with three levels, respectively. And the precision was also acceptable with relative standard deviation (RSD) below 20% for all analytes. The developed methods were applied for the determination and quantification of target compounds in surface water, sediment, wastewater, and sludge samples collected from two wastewater treatment plants (WWTPs) and the Pearl River in Guangzhou, China. BTHs were frequently detected in surface water and wastewater, while UV filters were mainly found in sediment and sludge. Benzotriazole (BT) and 2-hydroxybenzothiazole (2-OH-BTH) were the two major BTHs in influent wastewater and surface water, respectively, with concentrations up to 966 and 189 ng/L. As for sediment and sludge, 2-(2'-hydroxy-5'-octylphenyl)-benzotriazole (UV-329) was a predominant chemical, detected at concentrations of 111 and 151 ng/g, respectively.

GcvB Regulon Revealed by Transcriptomic and Proteomic Analysis in Vibrio alginolyticus.

Vibrio alginolyticus is a widely distributed marine bacterium that is a threat to the aquaculture industry as well as human health. Evidence has revealed critical roles for small RNAs (sRNAs) in bacterial physiology and cellular processes by modulating gene expression post-transcriptionally. GcvB is one of the most conserved sRNAs that is regarded as the master regulator of amino acid uptake and metabolism in a wide range of Gram-negative bacteria. However, little information about GcvB-mediated regulation in V. alginolyticus is available. Here we first characterized GcvB in V. alginolyticus ZJ-T and determined its regulon by integrated transcriptome and quantitative proteome analysis. Transcriptome analysis revealed 40 genes differentially expressed (DEGs) between wild-type ZJ-T and gcvB mutant ZJ-T-ΔgcvB, while proteome analysis identified 50 differentially expressed proteins (DEPs) between them, but only 4 of them displayed transcriptional differences, indicating that most DEPs are the result of post-transcriptional regulation of gcvB. Among the differently expressed proteins, 21 are supposed to be involved in amino acid biosynthesis and transport, and 11 are associated with type three secretion system (T3SS), suggesting that GcvB may play a role in the virulence besides amino acid metabolism. RNA-EMSA showed that Hfq binds to GcvB, which promotes its stability.

TIRPnet: Risk prediction of traditional Chinese medicine ingredients based on a deep neural network.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a history of over 3000 years of medical practice. Due to the complex ingredients and unclear pharmacological mechanism of TCM, it is very difficult to predict its risks. With the increase in the number and severity of spontaneous reports of adverse drug reactions (ADRs) of TCM, its safety has received widespread attention. AIM OF THE STUDY: In this study, we proposed a framework based on deep learning to predict the probability of adverse reactions caused by TCM ingredients and validated the model using real-world data. MATERIALS AND METHODS: The spontaneous reporting data from Jiangsu Province of China was selected as the research data, which included 72,561 ADR reports of TCMs. All the ingredients of these TCMs were collected from the medical website and correlated with the corresponding ADRs. Then, a risk prediction model was constructed based on a deep neural network (DNN), named TIRPnet. Based on one-hot encoded data, our model achieved the optimal performance by fine-tuning some hyperparameters. The ten most commonly used TCM ingredients and their ADRs were collected as the test set to evaluate their performance as objective criteria. RESULTS: TIRPnet was constructed as a 7-layer DNN. The experimental results showed that TIRPnet performs excellently in all indicators, with a sensitivity of 0.950, specificity of 0.995, accuracy of 0.994, precision of 0.708, and F1 of 0.811. CONCLUSIONS: The proposed TIRPnet owns the ability to predict the ADRs of a single TCM ingredient by learning a large number of TCM-related spontaneous reports, which can help doctors design safe prescriptions and provide technical support for the pharmacovigilance of TCM.

Comparison of the Radiographic and Clinical Outcome Changes in Patients With Spinal Deformity Before and After Stretching DanceExercise: A Systematic Review and Meta-Analysis.

OBJECTIVE: This study aimed to compare radiographic and clinical outcomes of scoliosis and thoracic hyperkyphosis before and after stretching-based exercises. METHODS: Embase, PubMed, Cochrane Library, Web of Science, and Scopus databases were comprehensively searched for relevant studies from their inception to June 2022. Radiographic outcomes, including the Cobb angle of the main curve and thoracic kyphosis, and clinical outcomes, including the angle of trunk rotation (ATR), chest expansion, Numeric Rating Scale (NRS), and Scoliosis Research Society-22 Patient Questionnaire (SRS-22), were extracted. Pooled and subgroup analyses were performed using random or fixed-effects models based on I2 heterogeneity. RESULTS: In total, 334 patients from ten studies, including 255 patients with scoliosis and 79 patients with thoracic hyperkyphosis, were included in the meta-analysis. After stretching, the pooled results showed that the Cobb angle of the main curve and thoracic kyphosis significantly decreased (P < 0.001) in patients with scoliosis, and thoracic kyphosis, respectively. Angle of trunk rotation (ATR) decreased significantly (P = 0.003), and chest expansion improved significantly (P = 0.04) after stretching-based exercise. In addition, our pooled results showed that the NRS score was significantly reduced (P < 0.001) and that the SRS-22 scores of mental health (P = 0.003) and self-perceived image (P < 0.001) were significantly increased after stretching. CONCLUSIONS: Partial correction can be achieved using stretching-based exercises. Moreover, stretching-based exercises can reduce pain in patients and improve their quality of life. However, the optimal duration required further elucidation.

CsrA Regulates Swarming Motility and Carbohydrate and Amino Acid Metabolism in Vibrio alginolyticus.

Vibrio alginolyticus, like other vibrio species, is a widely distributed marine bacterium that is able to outcompete other species in variable niches where diverse organic matters are supplied. However, it remains unclear how these cells sense and adjust metabolic flux in response to the changing environment. CsrA is a conserved RNA-binding protein that modulates critical cellular processes such as growth ability, central metabolism, virulence, and the stress response in gamma-proteobacteria. Here, we first characterize the csrA homolog in V. alginolyticus. The results show that CsrA activates swarming but not swimming motility, possibly by enhancing the expression of lateral flagellar associated genes. It is also revealed that CsrA modulates the carbon and nitrogen metabolism of V. alginolyticus, as evidenced by a change in the growth kinetics of various carbon and nitrogen sources when CsrA is altered. Quantitative RT-PCR shows that the transcripts of the genes encoding key enzymes involved in the TCA cycle and amino acid metabolism change significantly, which is probably due to the variation in mRNA stability given by CsrA binding. This may suggest that CsrA plays an important role in sensing and responding to environmental changes.

Molecular cloning, expression and functional analysis of NF-kB1 p105 from sea cucumber Holothuria leucospilota.

The nuclear factor-κB (NF-κB) family is evolutionary conserved and plays key roles in the regulation of numerous basic cellular processes. In this study, a sea cucumber Holothuria leucospilota NF-κB1 p105 named HLp105 was first obtained. The full-length cDNA of HLp105 is 6564 bp long, with a 219 bp 5' untranslated region (UTR), a 2979 bp 3' UTR, and a 3366 bp open reading frame (ORF) encoding for 1121 amino acids with a deduced molecular weight of 123.92 kDa and an estimated pI of 5.31. HLp105 protein contains the conserved domain RHD, IPT, ANK and DEATH. HLp105 mRNA can be detected in all tissues examined, with the highest level in the intestine, followed by the transverse vessel, rete mirabile, coelomocytes, respiratory tree, bolishiti, cuvierian tubules, body wall, oesophagus and muscle. Challenged by LPS or poly (I:C), the transcription level of HLp105 was apparently up-regulated in the tissues examined. Besides, Over-expression of HLp105 in HEK293T cells, the apoptosis was inhibited, and the cytokines IL-1ß and TNF-α were activated. The results are important for better understanding the function of NF-κB1 p105 in sea cucumber and reveal its involvement in immunoreaction.

A novel method for signal detection of adverse drug reactions based on proportional reporting ratios.

OBJECTIVE: The proportional reporting ratio (PRR) is a statistical method for signal detection of adverse drug reactions (ADRs) based on unbalanced proportions. Although effective, this method only takes into account the proportional relation based on target adverse reactions and ignores the relation between a given ADR and the others for the same drug. Therefore, it is necessary to improve the calculation deviation in PRR. In this study, we developed a novel PRR (NPRR) method and compared it with the original PRR method for the purpose of a combined application of these two methods for ADR signal detection. METHODS: NPRR is also based on unbalanced proportions, in which the proportion for a given ADR is linked to a specific drug (or all other drugs), and then divided by the corresponding proportion for all other ADRs. RESULTS: Applying this method to the ADR data of Jiangsu Province, China in 2008 and 2009, we detected 3,021 signals. Compared with the PRR method, the sensitivity of our method is 0.99, the specificity is 0.97, and the Youden index is 0.96. CONCLUSION: NPRR is an excellent method supplementary to PRR. The combination of these two methods can reduce calculation deviation and detect ADRs more effectively.

Risk Factors of Recurrent Ischemic Events after Acute Noncardiogenic Ischemic Stroke.

BACKGROUND: A considerable proportion of acute noncardiogenic ischemic stroke patients continue to experience recurrent ischemic events after standard therapy. AIM: We aimed to identify risk factors for recurrent ischemic event prediction at an early stage. METHODS: 286 non-cardioembolic ischemic stroke patients with the onset of symptoms within 24 hours were enrolled. Vascular risk factors, routine laboratory data on admission, thromboelastography test seven days after clopidogrel therapy and any recurrent events within one year were assessed. Patients were divided into case group (patients with clinical adverse events, including ischemic stokes, transient ischemic attack, myocardial infarction and vascular related mortality) and control group (events-free patients). The risk of the recurrent ischemic events was determined by the receiver operating characteristic curve and multivariable logistic regression analysis. RESULTS: Clinical adverse events were observed in 43 patients (case group). The mean levels of Mean Platelet Volume (MPV), Platelet/Lymphocyte Ratio (PLR), Lymphocyte Count (LY) and Fibrinogen (Fib) on admission were significantly higher in the case group as compared to the control group (P<0.001). Seven days after clopidogrel therapy, the ADP-induced platelet inhibition rate (ADP%) level was lower in the case group, while the Maximum Amplitude (MA) level was higher in the case group as compared to the control group (P<0.01). The Area Under the Curve (AUC) of receiver operating characteristic(ROC) curve of LY, PLR, , Fib, MA, ADP% and MPV were 0.602, 0.614, 0.629, 0.770, 0.800 and 0.808, respectively. The logistic regression analysis showed that MPV, ADP% and MA were indeed predictive factors. CONCLUSION: MPV, ADP% and MA were risk factors of recurrent ischemic events after acute noncardiogenic ischemic stroke. Urgent assessment and individual drug therapy should be offered to these patients as soon as possible.

Anticancer effects of curcumin on nude mice bearing lung cancer A549 cell subsets SP and NSP cells.

Curcumin is a key polyphenolic curcuminoid extracted from the root of turmeric rhizome Curcuma longa Linn, which is a frequently used Chinese herb for the treatment of cancer. The aim of the present study was to investigate the mechanism of the inhibitory effects of curcumin on nude mice with lung cancer A549 cell subsets side population (SP) and non-SP (NSP) cells. BALB/c mice were subcutaneously injected with the tumor cells of A549 SP or NSP subsets consisting of 1×109 cells/l (0.2 ml in total). After 16 days of inoculation with A549, the mice were intraperitoneally injected with curcumin (100 mg/kg, 0.2 ml) once every other day, eight times in total. A series of assays were performed to detect the effects of curcumin on: i) Tumor weight and size; ii) Notch and hypoxia inducible factor 1 (HIF-1) mRNA expression by quantitative polymerase chain reaction; and iii) vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) by immunohistochemistry. It was determined that curcumin decreased the tumor weight and size, downregulated the expression of Notch and HIF-1 mRNA and suppressed the VEGF and NF-κB expression. These results indicated that curcumin inhibited lung cancer growth through the regulation of angiogenesis mediated by VEGF signaling.

Two-stage fermentation for 2-Ketogluconic acid production by Klebsiella pneumoniae.

2-Ketogluconic acid production by Klebsiella pneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism.

IL-4 induces a suppressive IL-10-producing CD8+ T cell population via a Cdkn2a-dependent mechanism.

CD8(+) T cells play an important role in immune regulation and effective immune responses against tumor cells, viral infection, and intracellular pathogens. In this report, using tiger or 10BiT mice, we defined a population of IL-10-producing CD8(+) T cells that were induced by IL-4. These IL-10(+)CD8(+) T cells possessed a strong inhibitory effect on the CD4(+) T cell proliferation in an IL-10-dependent and cell contact-dependent fashion. In comparison with IL-10(-)CD8(+) T cells, IL-10(+)CD8(+) T cells expressed an array of Th2-like cytokines (IL-4, IL-5), perforin, and granzymes, as well as the cell cycle regulatory protein Cdkn2a. Interestingly, knockdown of cdkn2a using siRNA reduced IL-4-induced IL-10 production significantly. Furthermore, CD8(+) T cells from Cdkn2a(-/-) mice produced a significantly lower amount of IL-10, and the effect was limited to CD8(+) T cells but not observed in CD4(+) T cells and APCs. Finally, IL-10(+)CD8(+) T cells played a protective role in the TNBS-induced murine colitis model, indicating a critical role of this population of CD8(+) T cells in regulatory immune responses. Taken together, we have defined a population of IL-10-producing CD8(+) Tregs induced by IL-4 and mediated by Cdkn2a.