Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS

Progress of nanoparticle drug delivery system for the treatment of glioma

Gliomas are typical malignant brain tumours affecting a wide population worldwide. Operation, as the common treatment for gliomas, is always accompanied by postoperative drug chemotherapy, but cannot cure patients. The main challenges are chemotherapeutic drugs have low blood-brain barrier passage rate and a lot of serious adverse effects, meanwhile, they have difficulty targeting glioma issues. Nowadays, the emergence of nanoparticles (NPs) drug delivery systems (NDDS) has provided a new promising approach for the treatment of gliomas owing to their excellent biodegradability, high stability, good biocompatibility, low toxicity, and minimal adverse effects. Herein, we reviewed the types and delivery mechanisms of NPs currently used in gliomas, including passive and active brain targeting drug delivery. In particular, we primarily focused on various hopeful types of NPs (such as liposome, chitosan, ferritin, graphene oxide, silica nanoparticle, nanogel, neutrophil, and adeno-associated virus), and discussed their advantages, disadvantages, and progress in preclinical trials. Moreover, we outlined the clinical trials of NPs applied in gliomas. According to this review, we provide an outlook of the prospects of NDDS for treating gliomas and summarise some methods that can enhance the targeting specificity and safety of NPs, like surface modification and conjugating ligands and peptides. Although there are still some limitations of these NPs, NDDS will offer the potential for curing glioma patients

Thrombectomy versus combined thrombolysis and thrombectomy in patients with large vessel occlusion and chronic kidney disease

Background: Whether intravenous thrombolysis (IVT) should be bridged before mechanical thrombectomy (MT) remains uncertain in patients with large vessel occlusion (LVO) and chronic kidney disease (CKD). Methods: This research systematically enrolled every patient with both acute ischemic stroke (AIS) and CKD who received MT and fulfilled the criteria for IVT from January 2015 to December 2022. According to whether they underwent IVT, the patients were categorized into two cohorts: MT and combined IVT + MT. A binary logistic regression model was used to adjust for potential confounders, and propensity score matching analysis was used to assess the efficacy and safety of IVT in AIS patients with CKD who underwent MT. Results: A total number of 406 patients were ultimately included in this study, with 236 patients in the MT group and 170 in the combined group. After PSM, there were 170 patients in the MT group and 170 in the combined group, and the clinical characteristics between the two groups were well balanced. The MT + IVT group had better long-term functional outcomes than the MT group (35.9% versus 21.2%, P = 0.003) and more modified thrombolysis in cerebral infarction (mTICI) (2b-3) (94.1% versus 87.6%, P = 0.038), while no significant difference was found regarding symptomatic intracranial hemorrhage (sICH). In line with the results observed in the in the postmatched population, the logistic regression revealed that patients in the IVT + MT group demonstrated superior clinical outcomes (adjusted OR 0.440 [95% CI (0.267–0.726)], P = 0.001) in the prematched population. Conclusion: For LVO patients with CKD and indications for IVT, IVT bridging MT improves their prognosis compared with direct MT

The Mycobacterial DNA Methyltransferase HsdM Decreases Intrinsic Isoniazid Susceptibility

Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is a serious infectious disease worldwide. Multidrug-resistant TB (MDR-TB) remains a global problem, and the understanding of this resistance is incomplete. Studies suggested that DNA methylation promotes bacterial adaptability to antibiotic treatment, but the role of mycobacterial HsdM in drug susceptibility has not been explored. Here, we constructed an inactivated Mycobacterium bovis (BCG) strain, ΔhsdM. ΔhsdM shows growth advantages over wild-type BCG under isoniazid treatment and hypoxia-induced stress. Using high-precision PacBio single-molecule real-time sequencing to compare the ΔhsdM and BCG methylomes, we identified 219 methylated HsdM substrates. Bioinformatics analysis showed that most HsdM-modified genes were enriched in respiration- and energy-related pathways. qPCR showed that HsdM-modified genes directly affected their own transcription, indicating an altered redox regulation. The use of the latent Wayne model revealed that ΔhsdM had growth advantages over wild-type BCG and that HsdM regulated trcR mRNA levels, which may be crucial in regulating transition from latency to reactivation. We found that HsdM regulated corresponding transcription levels via gene methylation; thus, altering the mycobacterial redox status and decreasing the bacterial susceptibility to isoniazid, which is closely correlated with the redox status. Our results provide valuable insight into DNA methylation on drug susceptibility

Association between pro-inflammatory diet and liver cancer risk: a systematic review and meta-analysis

Abstract Objective: This systematic review aimed to investigate the association between dietary inflammatory potential and liver cancer to provide evidence regarding scientific dietary health education. Design: Systematic review and meta-analysis. Setting: A comprehensive literature review was conducted to identify case–control or cohort studies that involved dietary inflammation index (DII)/empirical dietary inflammation pattern (EDIP) and liver cancer in PubMed, EMBASE, Cochrane, and Web of Science databases. Using a combination of DII/EDIP and liver cancer as the search terms, the associations between DII/EDIP and liver cancer were then assessed. Participants: Three case–control studies and two cohort studies were brought into the meta-analysis, with 225 713 enrolled participants. Results: Meta-analysis of categorical variables showed that DII/EDIP in the highest category increased the risk of liver cancer compared to DII/EDIP in the lowest category (relative risk (RR) = 2·35; 95 % CI 1·77, 3·13; P = 0·000) and with low heterogeneity across studies (I2 = 40·8 %, P = 0·119). Meta-analysis of continuous variables showed that significant positive association between liver cancer and DII/EDIP scores (RR = 1·24; 95 % CI 1·09, 1·40; P = 0·001), and no heterogeneity (I² = 0·0 %, P = 0·471). Stratified according to the study design, there was a significant positive association between liver cancer and DII/EDIP scores in both cohort studies (RR = 2·16; 95 % CI 1·51, 3·07; P = 0·000) and case–control studies (RR = 2·75; 95 % CI 1·71, 4·41; P = 0·000). Conclusion: The higher the DII/EDIP score, the higher the risk of liver cancer. This finding may have prominent implications for the general population

Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance

<div><p>Tuberculosis, caused by the pathogen <i>Mycobacterium tuberculosis</i>, is a worldwide public health threat. <i>Mycobacterium tuberculosis</i> is capable of resisting various stresses in host cells, including high levels of ROS and copper ions. To better understand the resistance mechanisms of mycobacteria to copper, we generated a copper-resistant strain of <i>Mycobacterium smegmatis</i>, mc²155-Cu from the selection of copper sulfate treated-bacteria. The mc²155-Cu strain has a 5-fold higher resistance to copper sulfate and a 2-fold higher resistance to isoniazid (INH) than its parental strain mc²155, respectively. Quantitative proteomics was carried out to find differentially expressed proteins between mc²155 and mc²155-Cu. Among 345 differentially expressed proteins, copper-translocating P-type ATPase was up-regulated, while all other ABC transporters were down-regulated in mc²155-Cu, suggesting copper-translocating P-type ATPase plays a crucial role in copper resistance. Results also indicated that the down-regulation of metabolic enzymes and decreases in cellular NAD, FAD, mycothiol, and glutamine levels in mc²155-Cu were responsible for its slowing growth rate as compared to mc²155. Down-regulation of KatG2 expression in both protein and mRNA levels indicates the co-evolution of copper and INH resistance in copper resistance bacteria, and provides new evidence to understanding of the molecular mechanisms of survival of <i>mycobacteria</i> under stress conditions.</p></div

Gene ontology analysis of 345 differentially expressed proteins between <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain.

<p>(A) Classification based on protein functions; (B) classification based on the proteins-associated biological processes.</p

Growth curve and the susceptibility of <i>M</i>. <i>smegmatis</i> to copper and isoniazid.

<p>(a) The growth curve of <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain mc²155-Cu were measured in 7H9 media. Experiments were performed in triplicate. Squares, mc2155-Cu strains; circle, mc2155 strain; (b) The bacterial growth on 7H10 plates for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with CuSO₄ at different concentrations for 3 days, respectively. The panels show serial dilution (1:10) of mc²155 and mc²155-Cu. Diluted M. smegmatis cultures were spotted onto solid 7H10 media in the presence of CuSO₄ ranged from 0 to 500 μM. Images were taken after 3 days incubation at 37°C. Images stand for 3 independent experiments.; and (c) The bacterial survival rate for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with 0.1 mg/ml isoniazid.***<i>p</i><0.001; n = 3.</p

Schematic representation of co-evolution of Copper and INH Resistance in <i>M</i>. <i>smegmatis</i>.

<p>Copper treatment induces the up-regulation of copper translocating P-type ATPase for enhancing bacterial resistance to copper and the down regulation of katG and NAD for increasing the bacterial resistance to INH.</p

Functional Characterization of Sirtuin-like Protein in <i>Mycobacterium smegmatis</i>

Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a <i>Mycobacterium smegmatis</i> sirtuin, MSMEG_5175, was overexpressed in a <i>M. smegmatis</i> strain mc²155 to generate an MSMEG_5175-overexpression strain (mc²155-MS5175) in the present study. The physiological aspects of mc²155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc²155 containing empty pMV261 plasmid (mc²155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc²155-pMV261 and mc²155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc²155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc²155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc²155-MS5175 as compared to those in mc²155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria