Improvement of Renal Function After Bariatric Surgery: a Systematic Review and Meta-analysis.

BACKGROUND/OBJECTIVE: The effect of bariatric surgery in renal function varies and the postoperative benefit time point remains unclear. We aim to assess the changes of renal function after bariatric surgery (BS) in different postoperative periods and subgroups. METHODS: We searched the databases of PubMed and Cochrane from inception to December 14, 2020. Articles included in the study were drawn from all recipients of BS that provided assessments of renal function pre and post-surgery. Meta-analysis was performed to compare glomerular filtration rate (GFR), serum creatinine, albumin-to-creatinine ratio (ACR), and albuminuria before and after BS. RESULTS: The study included 49 articles involving 8515 patients. Compared with pre-operative renal function, the overall analysis showed that bariatric surgery significantly reduced serum creatinine levels, ACR, and albuminuria. There was significant increase of GFR in the CKD subgroup, yet a noticeable decrease in the hyperfiltration subgroup. The most significant improvement in GFR was seen 6-12 months after BS, while ACR dropped most dramatically 12-24 months after BS. CONCLUSIONS: Bariatric surgery can improve renal function in obese patients with kidney dysfunction, especially 1 year after surgery.

[Intragastric balloon for weight reduction: rationale, benefits, risks and indications].

Intragastric balloon (IGB) placement under endoscopy is a non-invasive method for weight loss.By placing a space-occupying balloon in the stomach, IGB treatment can achieve better effect of weight loss than medications.Herein we review the development of IGB, its effect on weight loss and the mechanism, and the eligible individuals for IGB treatment.We also examine the high-intensity postoperative management following IGB placement, which is important for maintaining long-term weight loss, and discuss the future development of IGB.The patients should understand that on the basis of ensuring a high safety, the weight-losing effect of IGB can be limited and relies heavily on postoperative management.Patients should make a decision on IGB placement after careful consideration of their own physical, economic, and psychological conditions, lifestyle and the line of work in addition to the indications of IGB.IGB placement combined with high-intensity postoperative management and active interventions of lifestyle and dietary habits help to achieve long-term effect of weight loss and improve obesity-related complications.

[Therapeutic effect of laparoscopic Roux-en-Y gastric bypass in non-obese patients with type 2 diabetes].

OBJECTIVE: To evaluate the effectiveness of laparoscopic Roux-en-Y gastric bypass (LRYGB) surgery for treatment of type 2 diabetes (T2D) in patients with a body mass index (BMI) < 27.5 kg/m2. METHODS: We retrospectively analyzed the data of patients who underwent LRYGB surgery from March, 2012 to June, 2018 in the General Hospital of Guangzhou Military Command and Jinshazhou Hospital of Guangzhou University of Chinese Medicine. The changes in the parameters of glucose metabolism and physical indicators of the patients in the first, second and third years after the surgery were analyzed in patients in low BMI group and high BMI group. RESULTS: All the 74 patients underwent LRYGB successfully without conversion to open surgery. One year after the surgery, fasting blood glucose (FBG), HbA1c, postprandial blood glucose, fasting insulin, HOMA-IR, fasting C-peptide, BMI, body weight and waistline were significantly improved compared with their preoperative values in low BMI group (P < 0.05). At 2 years after the operation, FBG, HbA1c, postprandial blood glucose, HOMA-IR, BMI, body weight and waistline were significantly improved compared with the preoperative values in low BMI group (P < 0.05). In the third year, FBG, HOMA-IR, fasting C-peptide, body weight and waistline were significantly improved compared with the preoperative values in low BMI group (P < 0.05). There was no significant difference in the parameters of glucose metabolism and islet function between low BMI group and high BMI group at different stages. No serious complications occurred in these patients after the surgery. CONCLUSIONS: LRYGB is effective for treatment of T2D in Chinese patients with a BMI < 27.5. After the surgery, the patient show reduced waistline without significant weight loss. The long-term results of the surgery still require further investigations with a larger samples and longer follow-up.

[Opportunities and challenges of endoscopic sleeve gastroplasty].

Obesity and its complications, such as type 2 diabetes, hypertension, hyperlipidemia, nonalcoholic fatty liver, are serious global public health problems. Endoscopic sleeve gastroplasty (ESG) can reduce the length and width of the stomach by simulating the anatomical structure of surgical sleeve gastrectomy to reduce the capacity of the stomach, and is safe and effective to reduce weight. ESG has the advantages of non- invasiveness, no gastrectomy, repeatability, simple operation, no incision scar, few complications, short hospital stay and quick postoperative recovery. As an intermediate means of medical treatment and surgery, ESG provides a new method for weight loss for obese patients who cannot tolerate or are unwilling to undergo surgery. Herein we trace the origin of ESG, analyze the unique advantages of ESG suture, explore the technical improvement in the development of ESG, and briefly describe the weight reduction effect of ESG and compare the curative effect of ESG with laparoscopic sleeve gastrectomy. ESG has undergone rapid development and maturity but also faces such challenges as the lack of established standard procedures, unclear weight reduction mechanism, and clarification of the indications for operation. Still, ESG is expected to become the mainstream technique for weight reduction.

Single-Anastomosis Duodenal Jejunal Bypass Improve Glucose Metabolism by Regulating Gut Microbiota and Short-Chain Fatty Acids in Goto-Kakisaki Rats.

In recent years, bariatric surgery has emerged as a promising treatment for type 2 diabetes. Bariatric surgery is known to cause alterations in the relative abundance and composition of gut microbiota, which may lead to alterations in the levels of Short-Chain Fatty Acids (SCFAs) that are produced during fermentation by gut microbes. However, little is known about the mechanism of improved glucose metabolism mediated by gut microbiota following bariatric surgery. The aim of our study was to explore whether changes in gut microbiota and in fecal SCFA could be detected following single-anastomosis duodenal jejunal bypass (DJB-sa) surgery, a type of bariatric surgery, and whether these alterations might be related to the improvement of glucose metabolism. To this end, we performed DJB-sa or SHAM surgery on Goto-Kakisaki (GK) rats. We then compared the glucose metabolism as well as changes in gut microbiota and SCFAs levels between both groups. Our results showed that DJB-sa surgery was associated with a significant decrease in fasting blood glucose (FBG), intraperitoneal glucose tolerance test (IPGTT), and fasting serum insulin (FSI). And, DJB-sa led to a change in the composition of gut microbiota including an increase in the relative abundance of SCFA-producing bacteria (Bifidobacterium and Subdoligranulum). Moreover, the levels of six SCFAs in feces, as well as the intestinal expression of SCFA receptors including G-protein-coupled receptor 41 (GPR41), G-protein-coupled receptor 43 (GPR43), and G-protein-coupled receptor 109A (GPR109A), and the expression of Glucagon-like peptide-1 (GLP-1) displayed a significant increase following DJB-sa compared with the Sham group. Thus, the gut microbiota may contribute to the improvement of glucose metabolism in type 2 diabetes following DJB-sa. In conclusion, our study shows that DJB-sa improves glucose metabolism by modulating gut microbiota and by increasing short-chain fatty acid production.

Duodenojejunal Bypass Plus Sleeve Gastrectomy Reduces Infiltration of Macrophages and Secretion of TNF-α in the Visceral White Adipose Tissue of Goto-Kakizaki Rats.

BACKGROUND: Current studies indicate that inflammation of white adipose tissue (WAT) is a pathogenic characteristic of insulin resistance. However, the significance of visceral WAT inflammation after bariatric surgery remains unclear. METHODS: Duodenojejunal bypass plus sleeve gastrectomy (DJB-SG) was performed on Goto-Kakisaki rats. Weight, fasting blood glucose (FBG), and homeostatic model assessment of insulin resistance (HOMA-IR) in the DJB-SG group were compared to those in a sham surgery (SHAM) group every 2 weeks. The results of an oral glucose tolerance test (OGTT) and the volume of visceral adipose tissue (Visc.Fat) were compared before and 8 weeks postsurgery. Eight weeks after surgery, the rats were sacrificed and visceral WAT collected from the greater omentum. Tumor necrosis factor-α (TNF-α) and cluster of differentiation 68 (CD68) expression in the WAT were evaluated in paraffin-embedded sections by immunohistochemistry. RESULTS: Compared with the SHAM group, the DJB-SG group demonstrated a significant reduction in weight, FBG, and HOMA-IR (P < 0.05), with elevation of insulin levels (P < 0.05) from 4 weeks after surgery. OGTT and the quantity of Visc.Fat were significantly reduced (P < 0.05) 8 weeks after surgery. Moreover, the expression of TNF-α and CD68 in the visceral white adipose tissue was significantly lower 8 weeks after surgery (P < 0.05). CONCLUSIONS: The DJB-SG model established in Goto-Kakisaki rats achieved anticipated efficacy. Reduced TNF-α-related inflammation in visceral WAT may result in improved insulin resistance.

[Efficacy of two bariatric surgeries in type 2 diabetic patients with a body mass index of 25-27.5].

OBJECTIVE: To evaluate the therapeutic effects of laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic duodenal-jejunal bypass with sleeve gastrectomy (SADJB-SG) in patients with type 2 diabetes mellitus and a low body mass index (BMI) of 25-27.5. METHODS: Thirty-one type 2 diabetic patients with a BMI of 25-27.5 underwent bariatric surgeries in the General Hospital of Guangzhou Military Command between August, 2013 and August, 2015. The patients receiving LRYGB (17 cases) and SADJB-SG (14 cases) were compared for physical indexes, glucose metabolism and of pancreatic islet function at 1 year after the surgeries. RESULTS: No mortality occurred in the patients after the operations. At 1 year after the operation, the patients in LRYGB group showed significant improvements in body weight, BMI, glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), oral glucose tolerance test 2 h (OGTT2h), C-peptide, fasting insulin (FINS), and postprandial 2 hour insulin (2 hPINS) (P<0.05); in SADJB-SG group, significant improvements were observed in the body weigh, BMI, HbA1c, FPG, OGTT2h, and FINS after the operation (P<0.05). The postoperative improvements in body weigh, BMI, HbA1c, FPG, OGTT2h, C-peptide, and 2hPINS were comparable between SADJB-SG group and LRYGB group (P>0.05), but the incidence of postoperative anastomotic ulcer was lower in SADJB-SG group. CONCLUSION: SADJB-SG and LRYGB produce similar therapeutic effects in type 2 diabetic patients with a low BMI, but SADJB-SG is associated with a low incidence of postoperative complications and is therefore more suitable in such patients.

Reduced inflammatory responses of follicular helper T cell promote the development of regulatory B cells after Roux-en-Y gastric bypass.

Bariatric surgery is currently the most effective strategy in treating severe obesity and its comorbidities, such as type 2 diabetes (T2D). However, the mechanism through which bariatric surgery mediates its benefits is not completely understood. Since obesity and T2D represent yet another inflammatory disease, and follicular helper T (Tfh) cells play important roles in inflammatory disorders, we investigated whether the Tfh activity was altered after Roux-en-Y gastric bypass (RYGB), one of the most common bariatric surgery procedures. We found that the Tfh cells after RYGB were not significantly changed in number, but presented altered cytokine secretion profile, including lower interferon (IFN)-γ, interleukin (IL)-2, IL-4, and IL-17 secretion. Tfh cells after RYGB also downregulated inducible co-stimulator and programmed death-1. Interestingly, after Tfh cell-naive B cell coculture, Tfh cells after RYGB secreted more IL-10 than autologous Tfh cells before RYGB. The frequencies of IL-10-expressing and transforming growth factor (TGF)-ß-expressing regulatory B cells after Tfh cell-naive B cell coculture were directly correlated with the frequency of IL-10-expressing Tfh cells. Depletion of IL-10 in the coculture, however, resulted in fewer regulatory B cells. Finally, patients with greater increase in IL-10-expressing Tfh cells presented further reductions in body mass index, glycaemia, and body fat percentage. Together, these data demonstrated that the Tfh cells after RYGB presented lower inflammatory status and secreted higher IL-10, through which these Tfh cells promoted the development of regulatory B cells. Higher IL-10-expressing Tfh cell level also predicted better patient response to RYGB.

B cells present skewed profile and lose the function of supporting T cell inflammation after Roux-en-Y gastric bypass.

Bariatric surgeries, including Roux-en-Y gastric bypass (RYGB) are currently the best treatment for obesity and obesity-related comorbidities, such as type 2 diabetes. However, the underlying mechanism of bariatric surgeries is not entirely understood. Further investigations are needed to improve the success rate and achieve sustained health benefits. Given that B cell dysregulation is a critical component of etiology in inflammatory diseases, whereas obesity and type 2 diabetes represent two major inflammatory disorders, we investigated the effect of RYGB on B cell inflammation. We found that B cells after RYGB presented significantly elevated frequency of interleukin (IL)-10-producing cells and reduced frequency of IL-6-producing cells compared to those before RYGB. When grouping B cell subsets into regulatory (secreting IL-10 and transforming growth factor beta [TGF-ß]) and effector (secreting IL-2, IL-4, IL-6, IL-12, interferon gamma [IFN-γ] and tumor necrosis factor alpha [TNF-α]) types, we found that after RYGB, the regulatory to effector B cell ratio was significantly increased. Function analyses showed that B cells before RYGB supported IL-17 secretion from T cells whereas these cells after RYGB lost such capacity. B cells after RYGB also gained the capacity to suppress T cell IFN-γ production through TGF-ß-mediated effects, a feature not present in B cells before RYGB. Interestingly, the regulatory to effector B cell ratio was directly associated with the reductions in obesity markers following RYGB, such as BMI and fat mass percentage. Together, these results demonstrated a potential mechanism through which RYGB promoted amelioration of obesity and type 2 diabetes.

Monocytes play different roles in stimulating T cells in obese diabetic individuals.

Type 2 diabetes (T2D) is a chronic metabolic disorder, which was also found to involve a series of inflammatory disorders, including accumulation of macrophages and T cells in the adipose tissue, increased proinflammatory cytokine production, shifting of macrophage composition toward M1-type, and skewing of peripheral blood T cells toward IL-17 productions. However, these studies were primarily conducted in obese mouse models and/or human subjects with higher BMI, and may not reflect the role of the immune system in non-obese T2D pathogenesis. Here, we examined T cell and monocyte cytokine expression and function in both non-obese and obese T2D patients. We found that IFN-g production by circulating T cells were increased in both non-obese and obese T2D subjects, while IL-17 is only upregulated in obese T2D subjects. Also, circulating monocytes from obese T2D subjects had significantly higher IL-6 production than their counterparts in non-obese T2D subjects. Moreover, monocytes from non-obese T2D subjects could support IFN-g but not IL-17 production in vitro, while that from obese T2D subjects supported both IFN-g and IL-17 production. Together, our results revealed that the role immune system plays in T2D pathogenesis is more complicated than previously thought, and is affected by the person's BMI.

Profiling peripheral microRNAs in obesity and type 2 diabetes mellitus.

Mechanisms of type 2 diabetes mellitus (T2DM) remain elusive, in which obesity (OB) is considered as one of the major risk factors for the disease. A microRNA (miRNA) is a small non-coding RNA molecule functioning in RNA silencing and post-transcriptional regulation of gene expression. It has been demonstrated that some miRNAs can exist in serum stably and is closely related to various diseases. The goal of our study was to identify whether the deregulation of serum miRNAs was associated with T2DM and obesity. Twenty-five subjects with T2DM2, 25 healthy controls, 25 subjects with obesity, and 25 subjects with T2DM combined with obesity were included in the study. A total of 536 miRNA serum samples from these four groups were studied by miRNA polymerase chain reaction (PCR) panels. Data showed that miR-152 and miR-17 were significantly elevated in the OB group, whereas miR-138 was significantly decreased in OB group when compared to controls, T2DM, or T2DM+obesity group. In addition, level of MiR-593 was significantly lower in T2DM group and T2DM+obesity group when compared with controls. Further analysis revealed that the four miRNAs can be used as potential biomarkers to distinguish obesity from T2DM, OB+T2DM, and healthy subjects. Our study is one of the pioneer studies showing the differences in peripheral miRNA level in obesity, T2DM and T2DM combined with obesity. The study results suggest the potential utility of miRNAs in the prediction for obesity and T2DM.

Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus.

An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and by different operators demonstrated that the assay was reproducible (coefficient of variation 4.7-18.5% month-to-month; 3.3-8.8% person-to-person). The VEEV ECL assay exhibited no cross-reactivity with two closely related alphaviruses or with 21 heterologous biological agents. A genetically biotinylated recombinant VEEV antibody, MA116SBP, was evaluated for utility for detection of rE2; although functional in the ECL assay, the LOD was two log units higher (100 ng/ml vs 1 ng/ml) using MA116SBP than when chemically biotinylated antibody was used. The ECL assay detected VEEV at the lowest LOD (highest sensitivity) hitherto reported in the published literature and ECL assay results were generated in ∼60 min compared to a 6-8h period required for ELISA. Results have demonstrated a sensitive, rapid, and fully automated ECL immunoassay for detection and identification of VEEV.

Competition between wild-type and a marked recombinant baculovirus (Spodoptera exigua nucleopolyhedrovirus) with enhanced speed of action in insect larvae.

Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6-12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findingssuggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems.

Aerosol and nasal delivery of vaccines and antiviral drugs against seasonal and pandemic influenza.

Influenza is primarily a respiratory tract infection involving the exacerbation and inflammation of the respiratory tract, which can progress to life-threatening pneumonia, hypercytokinemia, edema, acute lung injury, respiratory failure and death. Viral mutations and drug resistance are the leading challenges in influenza prevention and treatment. Aerosol inhalation provides rapid availability and sustained therapeutic levels of antiviral drugs in the respiratory tract, without causing a systemic burden to unaffected tissues and organs. Furthermore, aerosol delivery enhances the bioavailability of antiviral drugs with poor oral adsorption. Nasal spray delivery of vaccines provides a safe and needle-free means of vaccination, and contains live-attenuated virus that induces mucosal immunity and provides long-lasting immunity relative to injectable inactivated vaccines. Since influenza is a disease with respiratory clinical manifestations, specific delivery of antiviral drugs or vaccines to the respiratory tract may represent a safe and effective approach to combat influenza.

A novel approach to development of monoclonal antibodies using native antigen for immunization and recombinant antigen for screening.

The production of monoclonal antibodies (MAb) specific to microbes is rapidly growing. Finding an appropriate antigen to screen hybridoma clones has become increasingly important. However, the conventional method, in which the purified antigen from the microbe is routinely used for screening, cannot avoid selection of false positive hybridoma clones, since even highly purified antigen is found to be contaminated with some other proteins from the microbe. In this study, MAbs against anthrax protective antigen (PA), the central component of the three-part toxin secreted by Bacillus anthracis were developed using a pair of the roughly purified native PA as an immunogen and the recombinant PA as a screening antigen without any possibility of false selection, since the recombinant PA was produced by a gene engineering approach and impossible to be contaminated with any other proteins from B. anthracis. In total, nine stable hybridoma clones secreting anti-PA MAbs were developed. All of them had the same type of heavy and light chains, IgG1/kappa. The binding profiles for these anti-PA MAbs were investigated by ELISA. This novel approach to the development of MAbs should be applicable to the production of MAbs to other microbes, especially to those from which antigens can hardly be purified to a high degree.

Identification of AcMNPV EXON0 (ac141) domains required for efficient production of budded virus, dimerization and association with BV/ODV-C42 and FP25.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late gene exon0 (ac141) is required for the efficient production of budded virus (BV). EXON0 interacts with nucleopcapsid protein BV/ODV-C42 and FP25 and enables egress of nucleocapsids from the nucleus to the cytoplasm. This study examines the functional domains of EXON0 that play a role in BV production. Six putative domains of the 261 amino acid EXON0 were deleted and examined for functionality by determining their ability to rescue an AcMNPV exon0 knockout bacmid. Domain mapping results showed that all the six domains were required but deletion of the N-terminal acidic region and the leucine zipper domains had the greatest impact on BV production. Yeast 2-hybrid and co-immunoprecipitation demonstrated that EXON0 formed dimers. Point mutation analysis demonstrated that the leucine zipper was required for dimer formation and interaction with BV/ODV-C42 and FP25. The charged domain was also required for BV/ODV-C42 interaction.

Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac142 is a baculovirus core gene and encodes a protein previously shown to associate with occlusion-derived virus (ODV). To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac142 deletion virus (AcBAC(ac142KO-PH-GFP)). Fluorescence and light microscopy revealed that AcBAC(ac142KO-PH-GFP) exhibits a single-cell infection phenotype. Titration assays and Western blot confirmed that AcBAC(ac142KO-PH-GFP) is unable to produce budded virus (BV). However, viral DNA replication is unaffected and the development of occlusion bodies in AcBAC(ac142KO-PH-GFP)-transfected cells evidenced progression to very late phases of the viral infection. Western blot analysis showed that AC142 is expressed in the cytoplasm and nucleus throughout infection and that it is a structural component of BV and ODV which localizes to nucleocapsids. Electron microscopy indicates that ac142 is required for nucleocapsid envelopment to form ODV and their subsequent occlusion, a fundamental process to all baculoviruses.

Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004); however, the function or mechanism by which EXON0 affects BV production is unknown. In this study, we further elucidated the function of EXON0 by investigating the localization of EXON0 in infected Sf9 cells and in virions and by identifying interactions between EXON0 and other viral proteins. In addition, electron microscopy was used to study the cellular localization of nucleocapsids in cells transfected with an exon0 knockout (KO) virus. The results showed that EXON0 was localized to both the cytoplasm and the nuclei of infected Sf9 cells throughout the infection. Western blotting results also showed that EXON0 was purified along with BV and occlusion-derived virus (ODV). The fractionation of BV into the nucleocapsid and envelope components showed that EXON0 localized to the BV nucleocapsid. Yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy revealed that it interacted with nucleocapsid proteins FP25 and BV/ODV-C42. Cells transfected with the exon0 KO virus exhibited normally appearing nucleocapsids in the nuclei in numbers equal to those in the nuclei of cells transfected with the EXON0 repaired virus. In contrast, the numbers of nucleocapsids in the cytoplasm of cells transfected with the exon0 KO virus were significantly lower than those in the cytoplasm of cells transfected with the repaired virus. These results support the conclusion that EXON0 is required in the BV pathway for the efficient egress of nucleocapsids from the nucleus to the cytoplasm.

Tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system.

The use of insect cells has been highly successful for the expression of foreign proteins from baculoviruses or plasmid vectors. Here, we describe a tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system. The system includes the DEF domains of the spruce budworm (Choristoneura fumiferana) EcR (CfEcR) fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and the EF domains of mammalian Mus musculus retinoid X receptor (MmRXR) fused to the acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0. Using a GAL4 response element in reporter constructs, both transient and stable expression in insect lepidopteran cells showed that the chimeric MmRXR and CfEcR only activated the reporter genes in the presence of inducer; no gene expression was detectable in the absence of inducer. Characterization of heterogenous activation domains in insect cells showed that the AADs from Autographa californica multiple nucleopolyhedrovirus (MNPV) IE1 and Orgyia pseudotsugata MNPV IE0 consistently exhibited higher inducible levels than the archetype AAD from herpesvirus VP16 in insect cells. To confirm the tight regulation of this system the highly toxic protein, diphtheria toxin (DT), was used. In the absence of an inducer no cytotoxic effect was observed in insect cells that had been transiently transformed with DT expressing plasmids. This system will therefore be a very useful tool for biotechnology applications expressing highly toxic proteins in insect cells and for studying the functional genomics of insects and microorganisms that infect them.