Construction of a eukaryotic expression system with stable and secretory expression of mycobacterium tuberculosis 38 kDa protein.

The 38 kDa protein is a major antigen of mycobacterium tuberculosis and has been widely used in TB serodiagnosis, due to its highly sensitivity and specificity. Here we attempt to establish a production platform of recombinant 38 kDa protein in mammalian cells and to evaluate the potential value of 38 kDa protein in TB serodiagnosis. The 38 kDa gene is synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-38 kDa-eGFP was packaged, titered, and then transduced into HEK 293 T cells. Recombinant cell lines were selected by limiting dilution. Supernatants were collected and purified by HisTrapTM HP column. Western blot showed a molecular weight of approximate 38 kDa in cell supernatants as expected. ELISA assay confirmed the immunological specificity of the obtained protein in the presence of MTB-infected human serum samples. In all, we have obtained a stable cell line with long-term and robust expression of secretory MTB 38 kDa protein, which may provide a promising candidate antigen for the development of TB serological diagnosis.

Equivalent Fracture Patterns Demonstrate Poorer Postoperative Functional Outcomes Among Pronation-External Rotation IV Ankle Fractures.

BACKGROUND: Pronation-external rotation IV (PER IV) ankle fractures are relatively uncommon among rotational ankle fractures, but they are the most severe type. Although recent studies have shown satisfactory functional recovery in PER IV after surgical treatment, the different outcomes between fracture patterns and equivalent fracture patterns have not yet been evaluated. This study aims to compare short-term outcomes in PER IV ankle injuries between fracture patterns and equivalent fracture patterns. METHODS: This retrospective study was conducted at Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou, China, from July 2023 to October 2023. A total of 41 PER IV injuries from 2018 to 2022 were included and followed for at least one year. The American Orthopaedic Foot and Ankle Society (AOFAS) Ankle­Hindfoot Scale, Ankle Range of Motions (ROM), and Visual Analogue Scale (VAS) for pain scores were the main outcome measures. The rate of postoperative complications was the secondary outcome measure. Patient demographics were compared in PER IV fractures and PER IV ankle equivalent fractures. RESULTS: The mean follow-up time was 18.2 ± 4.2 (range, 12-24) months. Postoperative X-ray and CT scans showed a satisfactory reduction of the ankle joint and syndesmosis. No reduction loss of distal tibiofibular syndesmosis or ankle joints was found at the 12-month follow-up. The average AOFAS scores after one year in both groups were satisfactory (fracture group vs. fracture equivalent group, 96.72 ± 4.21 vs. 92.63 ± 5.36; P < 0.05). The average VAS scores after one year in both groups were satisfactory (fracture group vs. fracture equivalent group, 1.45 ± 2.01 vs. 1.38 ± 1.96; P > 0.05). The average ROM scores after one year in both groups were satisfactory (dorsiflexion, fracture group vs. fracture equivalent group, 15.21 ± 5.62 vs. 13.46 ± 4.35; P > 0.05; plantar flexion, fracture group vs. fracture equivalent group, 38.62 ± 9.68 vs. 42.32 ± 5.28; P > 0.05). CONCLUSION: For patients with PER-IV ankle injuries, the fracture mode had a better prognosis than the fracture equivalent mode.

Comparative analysis of internal fixation modalities for PER type IV ankle fractures: a finite element study.

BACKGROUND: There are many options for the surgical treatment of pronation external rotation (PER) type IV ankle fractures, including the use of fibular plates and screws, the aim of this study was to investigate the biomechanical stability and safety of different internal fixation methods for PER type IV ankle fractures via finite element analysis. METHODS: A three-dimensional finite element model of the ankle joint and the whole foot of a healthy 26-years-old adult male was established and validated based on computed tomography images of his lower limb, and a computer-aided design was used to produce a PER type IV ankle fracture and plate and screw model. Four different internal fixation modes were simulated, including a (all ankle fixation-utilizing a fibular plate and screws for comprehensive stabilization of the ankle), b (inferior tibiofibular joint fixation + all ankle fixation), c (inferior tibiofibular joint fixation + unfixed anterior ankle), and d (inferior tibiofibular joint fixation + unfixed anterior and posterior ankles). The results of the four different fixation methods were compared via finite element analysis, and the von Mises stresses. The displacements of the four different fixation methods were analyzed as the output indices. RESULTS: There were no significant differences between the results of using fibular plates and screws and the displacement of fracture breaks among the four internal fixation modalities. The von Mises stress in the tibiotalar joint, median ankle, posterior ankle, and anterior ankle was minimized in the working condition of d, d, b, and d respectively. The von Mises stress in the fibular plate and screws was minimized in the working condition of a. The von Mises stress in the distal fibula was minimized in the working condition of a. However, the stress was mainly concentrated at the attachment point of the inferior tibiofibular anterior ligament, and in the working condition with inferior tibiofibular joint fixation, the stress was significantly concentrated in the inferior tibiofibular joint screw in all the fibular plates and screws. CONCLUSIONS: The results of this study demonstrate the feasibility of using finite element analysis to compare the biomechanical stability and safety of four configurations of fibular plates and screws for treating PER type IV ankle fractures. All four modalities provided comparable biomechanical stability and safety, showing no significant differences. However, the current limitations of the finite element analysis methodology preclude specific clinical inferences. Further refinement of the methodology in future studies is necessary to enable reliable clinical applications.

Caecum Rupture Secondary to Iliac Crest Bone Graft: A Case Report.

Autogenous bone grafting is a common surgical method in orthopaedics. The anterior iliac crest is a common site for harvesting autologous bone grafts. There are many complications after iliac bone harvesting, and pain and discomfort at the donor site are the most common sequelae. However, intestinal rupture after iliac bone harvesting has not been reported. We report a case of caecum rupture in a 58-year-old male after harvesting bone from his iliac crest. After proper surgical repair, the patient was discharged from the ICU and his bowel function recovered. This serious complication of bone harvesting from the iliac crest prompted investigation of the technique of iliac crest harvesting and donor site reconstruction.

Prediction model of adjacent vertebral compression fractures after percutaneous kyphoplasty: a retrospective study.

OBJECTIVES: The purpose of this study was to develop a prediction model to assess the risk of adjacent vertebral compression fractures (AVCFs) after percutaneous kyphoplasty (PKP) surgery. DESIGN: A retrospective chart review. SETTING AND PARTICIPANTS: Patients were collected from the Quzhou People's Hospital, from March 2017 to May 2019. Patients were included if they suffered from osteoporotic vertebral compression fractures (OVCFs), underwent PKP surgery and were followed up for 2 years. INTERVENTIONS: None. METHODS: This was a retrospective cohort study of all PKP surgery procedures of the thoracic, lumbar and thoracolumbar (TL) spine that have been performed for OVCF from 1 March 2017 up to 1 May 2019. The least absolute shrinkage and selection operator (LASSO) regression model was used to optimise feature selection for the AVCF risk model. Multivariable logistic regression analysis was applied to build a predicting model incorporating the feature selected in the LASSO regression model. The C-index, calibration plot and decision curve analysis were applied to assess this model. RESULTS: Gender, age, the number of surgical vertebrae, cement volume, bone mineral density, diabetes, hypertension, bone cement leakage, duration of anti-osteoporosis treatment after surgery and TL junction were identified as predictors. The model displayed good discrimination with a C-index of 0.886 (95% CI 0.828-0.944) and good calibration. High C-index value of 0.833 could still be reached in the interval validation. Decision curve analysis showed that the AVCF nomogram was clinically useful when intervention was decided at the AVCF possibility threshold of 1%. CONCLUSIONS: This study developed a clinical prediction model to identify the risk factors for AVCF after PKP surgery, and this tool is of great value in sharing surgical decision-making among patients consulted before surgery. TRIAL REGISTRATION NUMBER: researchregistry7716.

EDIII-Fc induces protective immune responses against the Zika virus in mice and rhesus macaque.

Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Zika Virus Envelope Protein induces G2/M Cell Cycle Arrest and Apoptosis via an Intrinsic Cell Death Signaling Pathway in Neuroendocrine PC12 Cells.

Clinical evidence suggests that there exists a strong correlation between Zika virus (ZIKV) infection and abnormal development of the nervous system. However, the underlying mechanisms remain to be elusive. In this study, recombinant lentiviral vectors coding for ZIKV structural proteins and truncations (prM-Env, M-Env and Env) were transduced into PC12 cells. Envelope (Env) overexpression induced significant inhibition of proliferation and triggered G2/M cell cycle arrest and apoptosis in PC12 cells. Flow cytometry and western blot analysis showed that the apoptosis was associated with up-regulation of both p53 and p21Cip1/Waf1 and down-regulation of cyclin B1. Presence of aberrant nuclei clusters were confirmed by immunofluorescence staining analysis. The data indicate that overexpression of prM-Env, M-Env or Env led to apoptosis via an intrinsic cell death signaling pathway that is dependent on the activation of caspase-9 and caspase-3 and accompanied by an increased ratio of Bax to Bcl-2 in transduced PC12 cells. In summary, our results suggest that ZIKV Env protein causes apoptosis in PC12 cells via an intrinsic cell death signaling pathway, which may contribute to ZIKV-induced abnormal development of the nervous system.