Development of an HSV-1 production process involving serum-reduced culturing and bead-to-bead transfer.

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

Effects of proof-of-concept training on reducing work stress and anxiety of junior medical staff: a randomized controlled trial proof-of-concept.

BACKGROUND: Psychological stress and anxiety have seriously affected the ability of new clinicians to adapt and coordinate their clinical work. Traditional pre-job training is often not very good at assisting new recruits to regulate their emotional problems. METHODS: This study is a randomized controlled study. A total of 435 newly recruited clinicians participated in the study. 428 clinicians were randomized into a control group (n = 214) and an intervention group (n = 214). The control group conducted regular pre-job training. Doctors of the intervention group attend a themed course every two weeks on the basis of regular induction training. Their physiological status was evaluated by Perceived Stress Scale (PPS-10), Generalized Anxiety Scale (GAD-7) and Psychological Resilience Scale (CD-RISC-10) 3 months later. Participants in the intervention group received a training satisfaction questionnaire. RESULTS: After entering the clinic for 3 months, the PSS-10 and GAD-7 scores of the intervention group were significantly lower than that of the control group. Consistently, the CD-RISC-10 score of new clinicians who received proof-of-concept pre-job training was significantly higher than that of new doctors in the control group. CONCLUSION: New doctors received the proof-of-concept group experienced alleviation in stress and anxiety.

Inhibitory effect of pirfenidone on pulmonary fibrosis in patients with acute paraquat poisoning.

OBJECTIVE: To study the efficacy of pirfenidone (PFD) on patients with pulmonary fibrosis caused by acute paraquat (PQ) poisoning. METHODS: A total of 86 patients with pulmonary fibrosis caused by acute PQ poisoning admitted to our hospital were analyzed retrospectively. All of them successfully received the standard 21-day treatment based on "Taishan Consensus", and they were assigned to the PFD group or the NO-PFD group according to whether they received PFD treatment (at 200 mg/time, 3 times/day) for 6 months after discharge. The two groups were compared in effective treatment rate, mortality and incidence of adverse reactions such as liver and kidney function damage, pulmonary fibrosis-associated indexes, pulmonary function-associated indexes, and arterial blood gas indexes before and after therapy. RESULTS: The PFD group showed a notably higher effective treatment rate than the NO-PFD group (P<0.05). Additionally, the PFD group showed notably lower levels of serum hyaluronic acid (HA), laminin (LN), type IV collagen (CIV), and type III procollagen (PCIII), and notably higher levels of forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and FEV1/FVC than the NO-PFD group (all P<0.001), and the PFD group also showed significantly higher levels of arterial blood gas indexes including arterial partial pressure of oxygen (PaO2) and PaO2/inspired oxygen (FIO2) than the NO-PFD group (both P<0.001). Moreover, the Kaplan-Meier survival curves showed that the survival rate of the patients in PFD group was significantly higher than that in the NO-PFD group (P<0.05). CONCLUSION: With a high safety, PFD can effectively improve the treatment efficacy in patients with pulmonary fibrosis caused by acute PQ poisoning. PFD can improve the pulmonary function and arterial blood gas status of patients, without causing obvious liver and kidney damage.

Effects of acrylonitrile on apoptosis of rat cerebral nerve cells.

The brain, especially the hippocampus, is sensitive to damage caused by anoxic chemicals. In this study, we established a rat model of acrylonitrile poisoning with administration by gavage, aiming to determine the influence of acrylonitrile on rat cerebral nerve cells. Transmission electron microscopy observation and TdT-mediated dUTP nick-end labelling (TUNEL) staining were used to explore preliminarily the apoptotic changes of cerebral nerve cells. The pathogenesis revealed by transmission electron microscopy indicated that apoptosis in the control group was more serious than that of the exposure groups. The results of TUNEL staining showed the apoptotic rate was significantly higher in the control group than that of other exposure groups. All the results indicated that acrylonitrile can inhibit the apoptosis of rat cerebral nerve cells, which is closely related to its animal carcinogenicity.

Association between social determinants and the presence of essential hypertension in type 2 diabetes mellitus patients.

The purpose of this study was to analyse the association between social determinants and the presence of essential hypertension in type 2 diabetes mellitus patients in one rural county (Jintan) of China. A health survey of Jintan residents was conducted in 2013, applying a cluster sampling method. Three hundred and fifty-seven type 2 diabetes mellitus patients were selected as research subjects. Among the patients selected, essential hypertension prevalence was 45.4% (n=162). After univariate analyses and logistic regression analyses, an association was observed between the presence of essential hypertension in type 2 diabetes mellitus patients and marital status, described as 'others (unmarried, divorce, widower, etc.)', and participating in active physical activity. Both of these two significant variables were positively associated with the higher prevalence of essential hypertension in type 2 diabetes mellitus patients. In conclusion, the patients suffering from type 2 diabetes mellitus combined with essential hypertension often reported an association with more negative social determinants (with the exception of participation in physical activity). Further strengthening the comprehensive multi-disease management to control and reduce the prevalence of essential hypertension in type 2 diabetes mellitus patients is required.

A novel lysosome-targeted fluorogenic probe based on 5-triazole-quinoline for the rapid detection of hydrogen sulfide in living cells.

A fluorogenic probe based on the novel fluorophore 5-triazole-quinoline was developed for the detection of hydrogen sulfide, an endogenous signaling molecule associated with the development of various diseases. The lysosome-targeted probe Lyso-HS was synthesized via C-H direct azidation from 8-aminoquinoline; it was able to detect H2S in 1 min and exhibited excellent turn-on ability with 95-fold fluorescence enhancement based on a new fluorochrome. The high quenching efficiency was further verified using time-dependent density functional theory (TDDFT). The probe also exhibited high selectivity and a low detection limit (as low as 214.5 nM), which has practical applications for disease detection and monitoring.

The Inhibitory Effects of Cu(2+) on Exopalaemon carinicauda Arginine Kinase via Inhibition Kinetics and Molecular Dynamics Simulations.

We studied the Cu(2+)-mediated inhibition and aggregation of Exopalaemon carinicauda arginine kinase (ECAK). We found that Cu(2+) significantly inactivated ECAK activity and double-reciprocal kinetics demonstrated that Cu(2+) induced noncompetitive inhibition of arginine and ATP (IC50 = 2.27 ± 0.16 µM; K i for arginine = 13.53 ± 3.76; K i for ATP = 4.02 ± 0.56). Spectrofluorometry results showed that Cu(2+) induced ECAK tertiary structural changes including the exposure of hydrophobic surfaces that directly induced ECAK aggregation. The addition of osmolytes such as glycine and proline successfully blocked ECAK aggregation induced by Cu(2+) and recovered ECAK activity. We built a 3D structure for ECAK using the ECAK ORF gene sequence. Molecular dynamics (MD) and docking simulations between ECAK and Cu(2+) were conducted to elucidate the binding mechanisms. The results showed that Cu(2+) blocked the entrance to the ATP active site; these results are consistent with the experimental result that Cu(2+) induced ECAK inactivation. Since arginine kinase (AK) plays an important role in cellular energy metabolism in invertebrates, our study can provide new information about the effect of Cu(2+) on ECAK enzymatic function and unfolding, including aggregation, and the protective effects of osmolytes on ECAK folding to better understand the role of the invertebrate ECAK metabolic enzyme in marine environments.

Microbial community structures in mixed bacterial consortia for azo dye treatment under aerobic and anaerobic conditions.

Thirteen pure strains that possessed high methyl red (MR)-decolorizing ability were isolated from dye-contaminated water. Each isolate was identified by 16S rDNA sequencing. The results reveal that all of the isolated strains were facultative anaerobic bacteria. Two novel bacterial consortia (AE and AN), which could decolorize MR under aerobic and anaerobic conditions, respectively, were developed. Azo dye decolorization rate was significantly higher with the use of consortia compared to that with the use of individual strains. Both of the consortia can decolorize different azo dyes effectively in a short time, and tolerate MR with high concentrations. To provide further insight into the microbial diversity of the bacteria consortia under aerobic and anaerobic conditions, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were performed. PCR-DGGE profiles revealed that the microbial community had changed significantly with varying initial concentrations of MR. Phylogenetic analysis indicated that microbial populations in the aerobic compartment belong to Klebsiella, Buttiauxella and Bacillus, whereas Klebsiella, Escherichia, Bacillus and Clostridium were present in the anaerobic compartment. Klebsiella, which was the majority genus in both of the consortia, may play an important role in azo dye removal.