Attenuating RNA Viruses with Expanded Genetic Codes to Evoke Adjustable Immune Response in PylRS-tRNACUAPyl Transgenic Mice.

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a prototypical RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. After NAEK supplementation, EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and protected their neonatal offspring from lethal challenges similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNACUAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

Kinetic analysis of paramyxovirus-sialoglycan receptor interactions reveals virion motility.

Many viruses initiate infection by binding to sialoglycan receptors at the cell surface. Binding to such receptors comes at a cost, however, as the sheer abundance of sialoglycans e.g. in mucus, may immobilize virions to non-functional decoy receptors. As a solution, sialoglycan-binding as well as sialoglycan-cleavage activities are often present in these viruses, which for paramyxoviruses are combined in the hemagglutinin-neuraminidase (HN) protein. The dynamic interactions of sialoglycan-binding paramyxoviruses with their receptors are thought to be key determinants of species tropism, replication and pathogenesis. Here we used biolayer interferometry to perform kinetic analyses of receptor interactions of animal and human paramyxoviruses (Newcastle disease virus, Sendai virus, and human parainfluenza virus 3). We show that these viruses display strikingly different receptor interaction dynamics, which correlated with their receptor-binding and -cleavage activities and the presence of a second sialic acid binding site. Virion binding was followed by sialidase-driven release, during which virions cleaved sialoglycans until a virus-specific density was reached, which was largely independent of virion concentration. Sialidase-driven virion release was furthermore shown to be a cooperative process and to be affected by pH. We propose that paramyxoviruses display sialidase-driven virion motility on a receptor-coated surface, until a threshold receptor density is reached at which virions start to dissociate. Similar motility has previously been observed for influenza viruses and is likely to also apply to sialoglycan-interacting embecoviruses. Analysis of the balance between receptor-binding and -cleavage increases our understanding of host species tropism determinants and zoonotic potential of viruses.

Comparative study of differentiating human pluripotent stem cells into vascular smooth muscle cells in hydrogel-based culture methods.

Vascular smooth muscle cells (VSMCs), which provides structural integrity and regulates the diameter of vasculature, are of great potential for modeling vascular-associated diseases and tissue engineering. Here, we presented a detailed comparison of differentiating human pluripotent stem cells (hPSCs) into VSMCs (hPSCs-VSMCs) in four different culture methods, including 2-dimensional (2D) culture, 3-dimensional (3D) PNIPAAm-PEG hydrogel culture, 3-dimensional (3D) alginate hydrogel culture, and transferring 3-dimensional alginate hydrogel culture to 2-dimensional (2D) culture. Both hydrogel-based culture methods could mimic in vivo microenvironment to protect cells from shear force, and avoid cells agglomeration, resulting in the extremely high culture efficiency (e.g., high viability, high purity and high yield) compared with 2D culture. We demonstrated hPSC-VSMCs produced from hydrogel-based culture methods had better contractile phenotypes and the potential of vasculature formation. The transcriptome analysis showed the hPSC-VSMCs derived from hydrogel-based culture methods displayed more upregulated genes in vasculature development, angiogenesis and blood vessel development, extracellular matrix compared with 2D culture. Taken together, hPSC-VSMCs produced from hydrogel-based culture system could be applied in various biomedical fields, and further indicated the suitable development of alginate hydrogel for industrial production by taking all aspects into consideration.

Application of symptom-based mind mapping combined with PBL teaching method in emergency trauma standardized resident training in MDT model.

Explore the feasibility and effectiveness of accepting mind mapping combined with problem-based learning (PBL) teaching method in the standardized training of emergency surgery residents in the multi-disciplinary team (MDT) model of emergency trauma. Eighty-nine doctors under training who rotated in the Department of Emergency Surgery of the First Affiliated Hospital of Anhui Medical University from January 2021 to January 2022 were selected as the study subjects, and randomly divided into a group receiving mind mapping combined with PBL teaching and a group receiving traditional lecture-based learning teaching. Mini-clinical evaluation exercise (Mini-CEX), direct observation of procedural skills (DOPS), teaching adherence, and satisfaction assessments were completed at the time of discharge from the department. There were no significant differences between the observation and control group trainees in terms of gender, age, education, and entry grades. Both groups of doctors were better able to participate in their respective teaching modes and made significant progress. The participants in the observation group had significantly higher Mini-CEX, DOPS, and teaching satisfaction scores than the control group (P < .05). Under the MDT model of emergency trauma, the combination of mind mapping and PBL teaching can improve the comprehensive clinical ability of the trainees more than participating in the traditional lecture-based learning teaching, which is worth promoting and implementing in the clinical standardized training.

A Novel Epithelial-Mesenchymal Transition Gene Signature Correlated With Prognosis, and Immune Infiltration in Hepatocellular Carcinoma.

Background: Although many genes related to epithelial-mesenchymal transition (EMT) have been explored in hepatocellular carcinoma (HCC), their prognostic significance still needs further analysis. Methods: Differentially expressed EMT-related genes were obtained through the integrated analysis of 4 Gene expression omnibus (GEO) datasets. The univariate Cox regression and Lasso Cox regression models are utilized to determine the EMT-related gene signature. Based on the results of multivariate Cox regression, a predictive nomogram is established. Time-dependent ROC curve and calibration curve are used to show the distinguishing ability and consistency of the nomogram. Finally, we explored the correlation between EMT risk score and immune immunity. Results: We identified a nine EMT-related gene signature to predict the survival outcome of HCC patients. Based on the EMT risk score's median, HCC patients in each dataset were divided into high and low-risk groups. The survival outcomes of HCC patients in the high-risk group were significantly worse than those in the low-risk group. The prediction nomogram based on the EMT risk score has better distinguishing ability and consistency. High EMT risk score was related to immune infiltration. Conclusion: The nomogram based on the EMT risk score can reliably predict the survival outcome of HCC patients, thereby providing benefits for medical decisions.

Characterization of the metabolite of cabozantinib generated from liver microsomes and hepatocytes by ultra-high performance liquid chromatography coupled to quadrupole/orbitrap high resolution mass spectrometry.

Cabozantinib is a potent inhibitor of tyrosine kinase receptor that plays key role in tumor pathogenesis. Cabozantinib has been approved by U. S. Food and Drug Administration for the treatment of cancer. The present work was aimed to explore the in vitro metabolism of cabozantinib using liver microsomes and hepatocytes from animal species and humans through ultra-high performance liquid chromatography coupled to quadrupole/orbitrap high resolution mass spectrometer. The metabolites were characterized by their elemental compositions, MS and MS/MS spectra. As a result, a total of 26 metabolites were identified, and 15 metabolites were newly reported. Among these metabolites, M12 (oxidative defluorination), M19 and M22 (demethylation), M21 (hydroxylation) and M26 (N-oxygenation) were the major metabolites in all species. Our data revealed that cabozantinib was metabolized via the following pathways: oxidative defluorination, hydroxylation, amide hydrolysis, O-dealkylation, N-oxygenation, demethylation and glucuronidation. Human recombinant cytochrome P450 (CYP) enzyme analysis revealed that metabolism of cabozantinib was mainly catalyzed by CYP3A4, while other CYP enzymes played negligible role. The current study provided valuable metabolic data of cabozantinib from different animal species and humans, which would aid in safety and efficacy assessment.

Comparative Study of Human Pluripotent Stem Cell-Derived Endothelial Cells in Hydrogel-Based Culture Systems.

Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) are promising cell sources for drug discovery, tissue engineering, and studying or treating vascular diseases. However, hPSC-ECs derived from different culture methods display different phenotypes. Herein, we made a detailed comparative study of hPSC-ECs from three different culture systems (e.g., 2D, 3D PNIPAAm-PEG hydrogel, and 3D alginate hydrogel cultures) based on our previous reports. We expanded hPSCs and differentiated them into ECs in three culture systems. Both 3D hydrogel systems could mimic an in vivo physiologically relevant microenvironment to protect cells from shear force and prevent cell agglomeration, leading to a high culture efficiency and a high volumetric yield. We demonstrated that hPSC-ECs produced from both hydrogel systems had similar results as 2D-ECs. The transcriptome analysis showed that PEG-ECs and alginate-ECs displayed a functional phenotype due to their higher gene expressions in vasculature development, extracellular matrix, angiogenesis, and glycolysis, while 2D-ECs showed a proliferative phenotype due to their higher gene expressions in cell proliferation. Taken together, both PEG- and alginate-hydrogel systems will significantly advance the applications of hPSC-ECs in various biomedical fields.

Hypertonic saline resuscitation protects against kidney injury induced by severe burns in rats.

BACKGROUND: Proper fluid resuscitation can relieve visceral damage and improve survival in severely burned patients. This study compared the effectiveness of resuscitation with 400mEq/L hypertonic saline (HS) and sodium lactate Ringer's solution (LR) in rats with kidney injury caused by burn trauma. METHODS: Rats (Sprague-Dawley) underwent burn injury and were randomized into sham, LR, and HS groups. Samples from the kidney were assayed for water content ratio, histopathology, and oxidative stress (superoxide dismutase (SOD) and malondialdehyde (MDA)). Serum sodium, renal function (creatinine and cystatin (Cys)-C), and inflammatory response (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and high mobility group protein box (HMGB)-1) were also examined as serum markers. RESULTS: Hypertonic saline resuscitation reduced the renal water content ratio and improved renal histopathology caused by severe burns. This effect was accompanied by reductions in serum creatinine and Cys-C as well as TNF-α, IL-1ß, and HMGB1. Serum sodium concentration and SOD activity were increased, whereas MDA content was decreased in the kidney tissue of the HS group. CONCLUSIONS: The data indicate that 400mEq/L HS solution reduces hyponatremia and renal edema, inhibits the release of inflammatory mediators, and alleviates oxidative stress injury, thus protecting against kidney injury induced by severe burns.

200mM hypertonic saline resuscitation attenuates intestinal injury and inhibits p38 signaling in rats after severe burn trauma.

BACKGROUND: An overabundant discharge of inflammatory mediators plays a significant role in intestinal injury throughout the early stages of critical burns. The present study aims to explore the outcome of 200mM hypertonic saline (HS) resuscitation on the intestinal injury of critically burned rats. MATERIALS AND METHODS: Fifty-six Sprague-Dawley rats were randomized into three groups: sham group (group A), burn plus lactated Ringer's group (group B), and burn plus 200mM HS group (group C). Samples from the intestine were isolated and assayed for wet-weight-to-dry-weight (W/D) ratio, histopathology analyses, and p38 mitogen-activated protein kinase (MAPK) activity. Serum interleukin 1ß (IL-1ß) and high mobility group protein box 1 (HMGB1) concentrations were also examined. RESULTS: Initial resuscitation with 200mM Na+ HS significantly decreased the intestinal W/D ratio and improved intestinal histopathology caused by severe burn. HS resuscitation also inhibited the increase of serum IL-1ß and HMGB1 concentrations, and p38 MAPK activity in the intestine of critically burned rats. CONCLUSIONS: The overall findings of this study suggest that preliminary resuscitation with 200mM HS after severe thermal injury reduces intestinal edema, inhibits systemic inflammatory response, and attenuates intestinal p38 MAPK activation, thus reduces burns-induced intestinal injury.

Effect of 200 mEq/L Na+ hypertonic saline resuscitation on systemic inflammatory response and oxidative stress in severely burned rats.

BACKGROUND: Excessive release of inflammatory mediators and oxidative stress play important roles in the increased vascular permeability and systemic edema during the early stage of severe burn. This study investigates the effect of 200 mEq/L Na(+) hypertonic saline (HS) on systemic inflammatory response and oxidative stress in severely burned rats. MATERIALS AND METHODS: Sprague-Dawley rats were divided into three groups: sham group, burn plus lactated Ringer's group, and burn plus HS group. Lung edema was assessed in terms of wet-weight-to-dry-weight ratio. Tumor necrosis factor α and interleukin 6 concentrations in serum were examined by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells were isolated and the expression of p38 mitogen-activated protein kinase was determined by Western blot analysis. The lung and intestinal concentrations of malondialdehyde, an indicator of oxidative stress, were also measured. RESULTS: Resuscitation with 200 mEq/L Na(+) HS significantly decreased the lung wet-weight-to-dry-weight ratio and abolished hyponatremia induced by burn injury. HS treatment also prevented the increases of myeloperoxidase activity and malondialdehyde content in the lung and intestine of severely burned rats. However, there were no significant differences, either in serum tumor necrosis factor α and interleukin 6 concentrations or with respect to the p38 mitogen-activated protein kinase expression in peripheral blood mononuclear cells, between the burn plus lactated Ringer's group and burn plus HS group (P > 0.05). CONCLUSIONS: Initial resuscitation with 200 mEq/L Na(+) HS after severe burn injury decreases pulmonary edema, prevents hyponatremia, and attenuates oxidative stress, but is not capable of inhibiting the systemic inflammatory response.

Mechanisms of the protective effects of BMSCs promoted by TMDR with heparinized bFGF-incorporated stent in pig model of acute myocardial ischemia.

This study investigates the mechanism by which transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor incorporated degradable stent implantation (TMDRSI) enhanced effects of bone marrow mesenchymal stem cells (BMSCs) transplantation against acute ischemic myocardial injury. After the mid-third of left anterior descending artery was ligated, miniswine were divided into none-treatment group (control, n = 6), BMSCs implantation group (C, n = 6), TMDRSI group (TS, n = 6) and TMDRSI and BMSCs implantation group (TSC, n = 6). Two channels of 3.5 mm diameter were established by a self-made drill in the ischemic region, into which a stent was implanted for the TS and TSC groups. Autologous BMSCs were transplanted into the ischemic region in C group or around the channels in TSC group. Expression of von Willebrand factor, vascular endothelial growth factor, interleukin-1ß, transforming growth factor-ß(3) , cell proliferation and apoptosis, histological and morphological analyses, myocardial remodelling and cardiac function were evaluated at different time-points. Six weeks after the operation, the above indices were significantly improved in TSC group compared with others (P < 0.05), though C and TS groups also showed better results than the control group (P < 0.05). The new method was shown to have activated paracrine pathway of transplanted BMSCs, increased survival and differentiation of such cells, and enhanced effects of BMSCs transplantation on myocardial remodelling, which may provide a new strategy for cell therapies against acute ischemic myocardial injury.

[Coronary artery lumen diameter and bifurcation angle derived from CT coronary angiographic image in healthy people].

OBJECTIVE: To observe the coronary vessel lumen diameter and bifurcation angle in subjects with normal CT coronary angiography (CTCA) imaging. METHODS: 64-row CT coronary angiography imaging from 526 adult people with excellent image quality and normal vascular image were analyzed in this study. The lumen diameter from the origin to distal with 2 mm lumen of left main coronary artery (LM), anterior descending branch (LAD), left circumflex branch (LCX) and right coronary artery (RCA) was measured at 1 cm interval in CPR image. The vascular tapered ratio was calculated. The bifurcation angle in the diagonal branch, obtuse marginal branch, posterior descending branch, acute marginal branch as well as the origin diameter was also measured in VR image. RESULTS: The LAD average length was 13 cm and lumen diameter was 3.92 mm at origin and 2.10 mm at distal. The average decremented ratio of LAD was 7.7% (male 7.0%, female 8.4%). The maximal decremented ratio 8.0% - 10.0% occurred at 3 - 5 cm apart from the origin of LAD. The LCX average length was 13 cm and lumen diameter was 3.57 mm at origin and 2.10 mm at distal. The average decremented ratio of LCX was 9.7% (male 9.6%, female 9.7%). Lumen decremented ratio was less than 3.0% between origin and proximal 3 cm and 8.3% - 10.7% in the rest portion of the LCX. The RCA average length was 18 cm and lumen diameter was 3.97 mm at origin and 2.15 mm at distal. The average decremented ratio of RCA was 5.1% (male 4.9%, female 5.3%). The decremented ratio of RCR was less than 4.0% between origin and proximal 10 cm and 6.1% - 15.2% in the rest portion. The bifurcation angle was 50, 55, 66 and 76 degree for LAD with diagonal branch, LCX with obtuse marginal branch, RCA with posterior descending branch and RCA with obtuse marginal branch respectively. CONCLUSION: Coronary artery length, lumen diameter and decremented ratio as well as bifurcation angel could be identified in 64 row CTCA image in vivo. This information could help us to understand the in vivo coronary artery anatomy.