Intracellular localized heterogeneous protein franking by a transmembrane domain of GP64 is sufficient to be assembled on budded virions of Bombyx mori nucleopolyhedrovirus.

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SPΔn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SPΔh-c) was significantly enhanced by 35-40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12-26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

Risk factors of postoperative delirium following spine surgery: A meta-analysis of 50 cohort studies with 1.1 million participants.

Objectives: Postoperative delirium (POD) is considered to be a common complication of spine surgery. Although many studies have reported the risk factors associated with POD, the results remain unclear. Therefore, we performed a meta-analysis to identify risk factors for POD among patients following spinal surgery. Methods: We systematically searched the PubMed, Embase and the Cochrane Library for relevant articles published from 2006 to February 1, 2023 that reported risk factors associated with the incidence of POD among patients undergoing spinal surgery. The Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guidelines were followed, and random effects models were used to estimate pooled odds ratio (OR) estimates with 95 % confidence intervals (CIs) for each factor. The evidence from observational studies was classified according to Egger's P value, total sample size, and heterogeneity between studies. Results: Of 11,329 citations screened, 50 cohort studies involving 1,182,719 participants met the inclusion criteria. High-quality evidence indicated that POD was associated with hypertension, diabetes mellitus, cardiovascular disease, pulmonary disease, older age (>65 years), patients experiencing substance use disorder (take drug ≥1 month), cerebrovascular disease, kidney disease, neurological disorder, parkinsonism, cervical surgery, surgical site infection, postoperative fever, postoperative urinary tract infection, and admission to the intensive care unit (ICU). Moderate-quality evidence indicated that POD was associated with depression, American Society of Anesthesiologists (ASA) fitness grade (>II), blood transfusion, abnormal potassium, electrolyte disorder, length of stay, inability to ambulate and intravenous fluid volume. Conclusions: Conspicuous risk factors for POD were mainly patient- and surgery-related. These findings help clinicians identify high-risk patients with POD following spinal surgery and recognize the importance of early intervention.

Risk factors of epidural hematoma in patients undergoing spinal surgery: a meta-analysis of 29 cohort studies.

OBJECTIVE: The authors conducted this meta-analysis to identify risk factors for spinal epidural haematoma (SEH) among patients following spinal surgery. METHODS: The authors systematically searched Pub: Med, Embase, and the Cochrane Library for articles that reported risk factors associated with the development of SEH in patients undergoing spinal surgery from inception to 2 July 2022. The pooled odds ratio (OR) was estimated using a random-effects model for each investigated factor. The evidence of observational studies was classified as high quality (Class I), moderate quality (Class II or III) and low quality (Class IV) based on sample size, Egger's P value and between-study heterogeneity. In addition, subgroup analyses stratified by study baseline characteristics and leave-one-out sensitivity analyses were performed to explore the potential sources of heterogeneity and the stability of the results. RESULTS: Of 21 791 articles screened, 29 unique cohort studies comprising 150 252 patients were included in the data synthesis. Studies with high-quality evidence showed that older patients (≥60 years) (OR, 1.35; 95% CI, 1.03-1.77) were at higher risk for SEH. Studies with moderate-quality evidence suggested that patients with a BMI greater than or equal to 25 kg/m² (OR, 1.39; 95% CI, 1.10-1.76), hypertension (OR, 1.67; 95% CI, 1.28-2.17), and diabetes (OR, 1.25; 95% CI, 1.01-1.55) and those undergoing revision surgery (OR, 1.92; 95% CI, 1.15-3.25) and multilevel procedures (OR, 5.20; 95% CI, 2.89-9.37) were at higher risk for SEH. Meta-analysis revealed no association between tobacco use, operative time, anticoagulant use or American Society of Anesthesiologists (ASA) classification and SEH. CONCLUSIONS: Obvious risk factors for SEH include four patient-related risk factors, including older age, obesity, hypertension and diabetes, and two surgery-related risk factors, including revision surgery and multilevel procedures. These findings, however, must be interpreted with caution because most of these risk factors had small effect sizes. Nonetheless, they may help clinicians identify high-risk patients to improve prognosis.

Bombyx mori Nucleopolyhedrovirus Hijacks Multivesicular Body as an Alternative Envelopment Platform for Budded Virus Egress.

Baculovirus budded virus (BV) acquires its envelope and viral membrane fusion proteins from the plasma membrane (PM) of the host cell during the budding process. However, this classical BV egress pathway has been questioned because an intracellularly localized membrane fusion protein, SPΔnGP64 (glycoprotein 64 [GP64] lacking the signal peptide [SP] n region), was assembled into the envelope to generate infective BVs in our recent studies. Here, we identify an additional pathway for Bombyx mori nucleopolyhedrovirus (BmNPV) BV assembly and release that differs, in part, from the currently accepted model for the egress pathway of baculovirus. Electron microscopy showed that during infection, BmNPV-infected cells contained many newly formed multivesicular body (MVB)-like compartments that included mature virions at 30 h postinfection (p.i.). Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB endosome marker, and GP64, a BmNPV fusion glycoprotein. MVB fusion with the PM and the release of mature virions, together with naked nucleocapsids, were observed at the cell surface. Furthermore, MVB egress mediated the translocation of SPΔnGP64 to the PM, which induced cell-cell fusion until 36 h p.i. This BV egress pathway can be partially inhibited by U18666A incubation and RNA interference targeting MVB biogenesis genes. Our findings indicate that BmNPV BVs are enveloped and released through MVBs via the cellular exosomal pathway, which is a subordinate BV egress pathway that produces virions with relatively inferior infectivity. This scenario has significant implications for the elucidation of the BmNPV BV envelopment pathway. IMPORTANCE BmNPV is a severe pathogen that infects mainly Bombyx mori, a domesticated insect of economic importance, and accounts for approximately 15% of economic losses in sericulture. BV production plays a key role in systemic BmNPV infection of larvae. Despite the progress made in the functional gene studies of BmNPV, BmNPV BV egress is ill-understood. This study reports a previously unreported MVB envelopment pathway in BmNPV BV egress. To our knowledge, this is the first report of a baculovirus using dual BV egress pathways. This specific BV egress mechanism explains the cause of the non-PM-localized SPΔnGP64-rescued gp64-null bacmid infectivity, elucidating the reason underlying the retention of SP by BmNPV GP64. The data obtained elucidate an alternate molecular mechanism of baculovirus BV egress.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Infectious hematopoietic necrosis virus truncated G protein effect on survival, immune response, and disease resistance in rainbow trout.

The major antigenic protein of infectious hematopoietic necrosis virus (IHNV) is the surface glycoprotein G, which contains neutralizing epitopes that induce the production of immune neutralizing antibodies. In this study, the IHNV G gene sequence was truncated according to bioinformatics principles and then recombinantly expressed via an E. coli expression system. We then assessed the specific antibody immunoglobin M (IgM) levels of rainbow trout immunized with recombinant truncated G protein (emulsified with Freund's incomplete adjuvant), and showed that antibody IgM levels of immunized fish were significantly higher than in the control group (p < 0.01). The mRNA expression levels of interferon 1 (IFN1) and interleukin-8 (IL-8) were also up-regulated significantly (p < 0.01) in head kidneys and spleens of rainbow trout immunized with recombinant truncated G protein. Also, after challenge with wild-type IHNV HLJ-09 virus on Day 28, rainbow trout immunized with recombinant truncated G protein showed cumulative survival rates of 60%. These results indicate that the truncated G protein of IHNV expressed by the E. coli prokaryotic expression system can be used as a candidate immunogen for an IHNV subunit vaccine, which lays a theoretical foundation for the study of further potential IHNV subunit vaccines.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in recruiting the ESCRT pathway through three ways in the host cells of fish during IHNV budding.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

[Overview of risk factors for failed percutaneous transforaminal endoscopic discectomy in lumbar disc herniation].

The lumbar disc herniation is a common and recurrent disease in the department of orthopedics. At present, the treatment means mainly include conservative treatment and surgical treatment. Compared with traditional open surgery, percutaneous transforaminal endoscopic discectomy (PTED) is safe, effective, economical and minimally invasive. It is widely used in minimally invasive treatment of lumbar disc herniation. However, the clinical reports of the failure of PTED are also common. According to the research reports of domestic and foreign scholars, there are varieties of risk factors for surgical failures, including the selections of patients, indications, surgical approaches and anesthesia methods preoperative. Occurrences of surgical complications including infection or left pains, and reasonable rehabilitation exercise after the operation are related to failures. There is no unified conclusion at present. In this paper, we reviewed the literatures about failed PTED, and try to make an overview about the general situation of failed operation in clinical practice, the risk factors for failures and the countermeasures.

Identification and subcellular location of an RNA silencing suppressor encoded by mulberry crinkle leaf virus.

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus recently identified from the woody plant mulberry (Morus alba L.). Little is known about the functions of the proteins encoded by the MCLV genome. Here, all the MCLV-encoded proteins were examined for the ability to suppress gene silencing by an agroinfiltration assay in combination with northern blot analysis of green fluorescent protein (GFP) mRNA and western blot analysis. Of the six proteins, only one protein, V3, which has been predicted to play a role in viral movement, was found to suppress the gene silencing induced by a sense GFP gene in Nicotiana benthamiana 16c. The minimal amino acid sequence of V3 that maintains suppressor activity was also determined by constructing truncated mutants lacking different lengths of the amino acid sequences at the N- or C-terminus of the V3 protein. The results showed that the 94 N-terminal amino acid residues of V3 are sufficient to maintain V3 suppressor activity. In addition, the subcellular location of the V3 protein was investigated by confocal laser scanning microscopy after the expression of a V3-RFP fused protein in leaf epidermal cells of N. benthamiana. The results indicated that the V3 protein localized not only to the cytoplasm but also to the nucleus of N. benthamiana, implying that V3 can shuttle between the nucleus and the cytoplasm. Deletion mutant analysis indicated that a putative nuclear localization signal (NLS) between aa 118-134 might be responsible for the nuclear distribution of the V3 protein. Given the importance of RNA silencing in plant-virus interactions, the identification of a silencing suppressor of MCLV should be valuable in understanding the pathogenicity and molecular biology of this virus.

[Application of plasma prostate electrovaporization system in the treatment of rectal cicatricial stenosis].

OBJECTIVE: To evaluate the feasibility, maneuver and efficacy of plasma prostate electrovaporization system in the treatment of rectal cicatricial stenosis. METHODS: According to similar procedure of transurethral resection prostate(TURP), intrarectal cicatriclectomy was performed with plasma prostate electrovaporization system in 7 patients with rectal low cicatricial stenosis after rectal cancer treatment (5 patients with transabdominal low anterior resection,2 patients with 3-dimension precise radiotherapy) to remove obstruction and dilate enteric cavity. RESULTS: Seven patients underwent 12 operations, including one operation in 3 patients, two operations in 3 patients, 3 operations in one patient. Resected rectal cicatricial tissue ranged from 5 to 15 g. Mean operation time was 41 min (25 to 40). Operation successful rate was 100% without complications such as perforation, bleeding and infection. All the patients had smooth defecation. CONCLUSION: Plasma prostate electrovaporization system is an effective treatment for rectal cicatricial stenosis with tiny trauma.