Staphylococcal Enterotoxin C2 Mutant-Induced Antitumor Immune Response Is Controlled by CDC42/MLC2-Mediated Tumor Cell Stiffness.

As a biological macromolecule, the superantigen staphylococcal enterotoxin C2 (SEC2) is one of the most potent known T-cell activators, and it induces massive cytotoxic granule production. With this property, SEC2 and its mutants are widely regarded as immunomodulating agents for cancer therapy. In a previous study, we constructed an MHC-II-independent mutant of SEC2, named ST-4, which exhibits enhanced immunocyte stimulation and antitumor activity. However, tumor cells have different degrees of sensitivity to SEC2/ST-4. The mechanisms of immune resistance to SEs in cancer cells have not been investigated. Herein, we show that ST-4 could activate more powerful human lymphocyte granule-based cytotoxicity than SEC2. The results of RNA-seq and atomic force microscopy (AFM) analysis showed that, compared with SKOV3 cells, the softer ES-2 cells could escape from SEC2/ST-4-induced cytotoxic T-cell-mediated apoptosis by regulating cell softness through the CDC42/MLC2 pathway. Conversely, after enhancing the stiffness of cancer cells by a nonmuscle myosin-II-specific inhibitor, SEC2/ST-4 exhibited a significant antitumor effect against ES-2 cells by promoting perforin-dependent apoptosis and the S-phase arrest. Taken together, these data suggest that cell stiffness could be a key factor of resistance to SEs in ovarian cancer, and our findings may provide new insight for SE-based tumor immunotherapy.

A Novel Pathway of Chlorimuron-Ethyl Biodegradation by Chenggangzhangella methanolivorans Strain CHL1 and Its Molecular Mechanisms.

Chlorimuron-ethyl is a widely used herbicide in agriculture. However, uncontrolled chlorimuron-ethyl application causes serious environmental problems. Chlorimuron-ethyl can be effectively degraded by microbes, but the underlying molecular mechanisms are not fully understood. In this study, we identified the possible pathways and key genes involved in chlorimuron-ethyl degradation by the Chenggangzhangella methanolivorans strain CHL1, a Methylocystaceae strain with the ability to degrade sulfonylurea herbicides. Using a metabolomics method, eight intermediate degradation products were identified, and three pathways, including a novel pyrimidine-ring-opening pathway, were found to be involved in chlorimuron-ethyl degradation by strain CHL1. Transcriptome sequencing indicated that three genes (atzF, atzD, and cysJ) are involved in chlorimuron-ethyl degradation by strain CHL1. The gene knock-out and complementation techniques allowed for the functions of the three genes to be identified, and the enzymes involved in the different steps of chlorimuron-ethyl degradation pathways were preliminary predicted. The results reveal a previously unreported pathway and the key genes of chlorimuron-ethyl degradation by strain CHL1, which have implications for attempts to enrich the biodegradation mechanism of sulfonylurea herbicides and to construct engineered bacteria in order to remove sulfonylurea herbicide residues from environmental media.

Continuous Oral Administration of the Superantigen Staphylococcal Enterotoxin C2 Activates Intestinal Immunity and Modulates the Gut Microbiota in Mice.

Staphylococcal Enterotoxin C2 (SEC2), a classical superantigen, is an antitumor immunotherapy agent. However, the injectable formulation of SEC2 limits its clinical application. Here, it is reported that oral administration of SEC2 activates the intestinal immune system and benefits intestinal health in a mouse model. These results indicate that intact SEC2 is detected in the stomach, intestine, and serum after oral administration. Continuous oral administration of SEC2 activates immune cells in gut-associated lymphoid tissues, promoting extensive differentiation and proliferation of CD4+ and CD8+ T cells and CD19+ B cells, leading to increased production of cytokines and secretory immunoglobulin A. SEC2 also enhances intestinal barrier function, as demonstrated by an increased villus length/crypt depth ratio and elevated expression of mucins and tight junction proteins. Additionally, SEC2 indirectly influenced gut microbiota, reinforcing potential probiotics and short-chain fatty acid synthesis. Enhanced differentiation of T and B cells in the spleen, coupled with elevated serum interleukin-2 levels, suggests systemic immune enhancement following oral administration of SEC2. These findings provide a scientific basis for the development of SEC2 as an oral immunostimulant for immune enhancement and anti-tumor immunotherapy.

Continuous oral exposure to micro- and nanoplastics induced gut microbiota dysbiosis, intestinal barrier and immune dysfunction in adult mice.

Micro/nanoplastics in the environment can be ingested by organisms and spread throughout the food chain, ultimately posing a threat to human health. However, the risk of continuous oral exposure in mammals remains unresolved. In this study, we utilized a continuous gavage mouse model to investigate the potential intestinal risks associated with oral exposure to polystyrene micro/nanoplastics (PS-MNPs) with environmentally relevant concentrations. The effects of PS-MNPs with different particle sizes on the gut microbiota, intestinal barrier, and intestinal immune function were evaluated. PS-MNPs can accumulate in the intestine after oral exposure and alter the composition of the gut microbiota. Exposure to PS-MNPs significantly reduced the ratio of Firmicutes to Bacteroidetes as well as the number of potentially beneficial bacteria in the gut, while the number of potentially harmful bacteria significantly increased. The short-chain fatty acids metabolized by gut microbiota were significantly changed by PS-MNPs. Exposure to PS-MNPs disrupts the function of the intestinal barrier and leads to inflammation in the intestines. The levels of secretory immunoglobulin A in the intestine and the differentiation of CD4+ and CD8+ T cells in mesenteric lymph nodes were significantly decreased by PS-MNPs. Moreover, the impact of PS-MNPs on mammalian intestinal health is influenced by the exposure duration and particle size, rather than the concentration. It also suggests that nanoplastics may pose more severe environmental risks.

[Growth and degradation characteristics of an efficient and broad-spectrum atrazine-degrading strain SB5]. / ä¸€æ ªé«˜æ•ˆå¹¿è°±èŽ åŽ»æ´¥é™è§£èŒSB5的生长和降解特性.

In this study, triazine-degrading strain SB5 was isolated and screened from the activated sludge contaminated with atrazine by enrichment culture technology. Based on its morphology and 16S rRNA gene analysis, strain SB5 was initially identified as Paenarthrobacter sp. It contained the atrazine-degrading genes trzN, atzB, and atzC. The addition of glucose, sucrose, sodium citrate, yeast extract and peptone to the culture medium significantly increased the biomass and atrazine degradation efficiency of strain SB5. The addition of (NH4)2SO4 and NH4Cl inhibited the biomass of strain SB5, but did not affect its degradation efficiency for atrazine. The addition of starch did not affect the biomass of strain SB5, but significantly inhibited its degradation for atrazine. Strain SB5 showed good atrazine tolerance and atrazine degradation ability in the temperature range of 4-42 ℃, initial pH of 4-10 and initial concentration of 50-1000 mg·L-1. Using 100 mg·L-1 atrazine as the sole carbon source, the strain SB5 degraded 100% of atrazine within 36 h under the optimal conditions of 37 ℃ and initial pH 8.0. The results of degradation spectrum analysis showed that strain SB5 had a good degradation effect on the six triazine herbicides (simazine, terbuthylazine, propazine, cyanazine, ametryn and prometryn) at an initial concentration of 100 mg·L-1, and the degradation rates were 86.4%, 92%, 98.6%, 95.6%, 100% and 99.2% after 48 h of incubation, respectively. The results demonstrated that SB5 was an efficient and broad-spectrum degradation strain. The strain SB5 further enriched the strain resources for atrazine biodegradation, and its high-efficient and broad-spectrum degradation characteristics for triazine herbicides showed a potential application value in the development of bioremediation technology for the pollution of triazine herbicides.

Toxic effects of nanoplastics with different sizes and surface charges on epithelial-to-mesenchymal transition in A549 cells and the potential toxicological mechanism.

As a newly emerging hazardous material, airborne nanoplastics are easily inhaled and accumulated in human and animal alveoli. We previously found that polystyrene nanoplastics (PS-NPs) induced apoptosis and inflammation of human alveolar epithelial A549 cells, implying they increase the risk of pulmonary fibrosis. In this study, we investigated whether PS-NPs induce epithelial-to-mesenchymal transition (EMT), the prelude to lung fibrosis, in A549 cells. A549 cells treated with PS-NPs of different sizes and surface charges exhibited increased migration and EMT markers accompanied with up-regulation of reactive oxygen species (ROS) and NADPH oxidase 4 (NOX4), an ROS generator located in the mitochondria and endoplasmic reticulum (ER). Moreover, PS-NPs caused mitochondrial dysfunction as demonstrated by membrane potential changes and impaired cellular energy metabolism. PS-NPs also activated ER stress as indicated by the up-regulated ER stress markers. As expected, smaller PS-NPs with a positive surface charge had stronger effects. Furthermore, the effects of PS-NPs on A549 cells were reversed by NOX4 gene knock-down, which verified the involvement of NOX4. Our results suggest that PS-NPs induce EMT in A549 cells through multiple mechanisms, and NOX4 is a key mediator in this process. Our findings contribute to understanding the toxicological mechanisms of nanoplastics on the respiratory system.

In vitro study on the toxicity of nanoplastics with different charges to murine splenic lymphocytes.

Nanoplastics can be ingested by organisms and penetrate biological barriers to affect multiple physiological functions. However, few studies have focused on the effects of nanoplastics on the mammalian immune system. We evaluated the effects and underlying mechanism of nanoplastics of varying particle sizes and surface charges on murine splenic lymphocytes. We found that nanoplastics penetrated into splenic lymphocytes and that nanoplastics of a diameter of 50 nm were absorbed more efficiently by the cells. The nanoplastics decreased cell viability, induce cell apoptosis, up-regulated apoptosis-related protein expression, elicited the production of reactive oxygen species, altered mitochondrial membrane potential, and impaired mitochondrial function. Positively charged nanoplastics exerted the strongest toxicity. Negatively charged and uncharged nanoplastics caused oxidative stress and mitochondrial structural damage in lymphocytes, while positively charged nanoplastics induced endogenous apoptosis directly. Moreover, nanoplastics inhibited the expression of activated T cell markers on the T cell surface, while inhibiting the differentiation of CD8+ T cells and the expression of helper T cell cytokines. In terms of the mechanism, a series of key signaling molecules in the pathways of T cell activation and function were markedly down-regulated after exposure to nanoplastics.

Characterizing the Microbial Consortium L1 Capable of Efficiently Degrading Chlorimuron-Ethyl via Metagenome Combining 16S rDNA Sequencing.

Excessive application of the herbicide chlorimuron-ethyl (CE) severely harms subsequent crops and poses severe risks to environmental health. Therefore, methods for efficiently decreasing and eliminating CE residues are urgently needed. Microbial consortia show potential for bioremediation due to their strong metabolic complementarity and synthesis. In this study, a microbial consortium entitled L1 was enriched from soil contaminated with CE by a "top-down" synthetic biology strategy. The consortium could degrade 98.04% of 100 mg L-1 CE within 6 days. We characterized it from the samples at four time points during the degradation process and a sample without degradation activity via metagenome and 16S rDNA sequencing. The results revealed 39 genera in consortium L1, among which Methyloversatilis (34.31%), Starkeya (28.60%), and Pseudoxanthomonas (7.01%) showed relatively high abundances. Temporal succession and the loss of degradability did not alter the diversity and community composition of L1 but changed the community structure. Taxon-functional contribution analysis predicted that glutathione transferase [EC 2.5.1.18], urease [EC 3.5.1.5], and allophanate hydrolase [EC 3.5.1.54] are relevant for the degradation of CE and that Methyloversatilis, Pseudoxanthomonas, Methylopila, Hyphomicrobium, Stenotrophomonas, and Sphingomonas were the main degrading genera. The degradation pathway of CE by L1 may involve cleavage of the CE carbamide bridge to produce 2-amino-4-chloro-6-methoxypyrimidine and ethyl o-sulfonamide benzoate. The results of network analysis indicated close interactions, cross-feeding, and co-metabolic relationships between strains in the consortium, and most of the above six degrading genera were keystone taxa in the network. Additionally, the degradation of CE by L1 required not only "functional bacteria" with degradation capacity but also "auxiliary bacteria" without degradation capacity but that indirectly facilitate/inhibit the degradation process; however, the abundance of "auxiliary bacteria" should be controlled in an appropriate range. These findings improve the understanding of the synergistic effects of degrading bacterial consortia, which will provide insight for isolating degrading bacterial resources and constructing artificial efficient bacterial consortia. Furthermore, our results provide a new route for pollution control and biodegradation of sulfonylurea herbicides.

Whole-Genome Sequencing of a Chlorimuron-Ethyl-Degrading Strain: Chenggangzhangella methanolivorans CHL1 and Its Degrading Enzymes.

Chlorimuron-ethyl is a commonly used sulfonylurea herbicide, and its long-term residues cause serious environmental problems. Biodegradation of chlorimuron-ethyl is effective and feasible, and many degrading strains have been obtained, but still, the genes and enzymes involved in this degradation are often unclear. In this study, whole-genome sequencing was performed on chlorimuron-ethyl-degrading strain, Chenggangzhangella methanolivorans CHL1. The complete genome of strain CHL1 contains one circular chromosome of 5,542,510 bp and a G+C content of 68.17 mol%. Three genes, sulE, pnbA, and gst, were predicted to be involved in the degradation of chlorimuron-ethyl, and this was confirmed by gene knockout and gene complementation experiments. The three genes were cloned and expressed in Escherichia coli BL21 (DE3) to allow for the evaluation of the catalytic activities of the respective enzymes. The glutathione-S-transferase (GST) catalyzes the cleavage of the sulfonylurea bridge of chlorimuron-ethyl, and the esterases, PnbA and SulE, both de-esterify it. This study identifies three key functional genes of strain CHL1 that are involved in the degradation of chlorimuron-ethyl and also provides new approaches by which to construct engineered bacteria for the bioremediation of environments polluted with sulfonylurea herbicides. IMPORTANCE Chlorimuron-ethyl is a commonly used sulfonylurea herbicide, worldwide. However, its residues in soil and water have a potent toxicity toward sensitive crops and other organisms, such as microbes and aquatic algae, and this causes serious problems for the environment. Microbial degradation has been demonstrated to be a feasible and promising strategy by which to eliminate xenobiotics from the environment. Many chlorimuron-ethyl-degrading microorganisms have been reported, but few studies have investigated the genes and enzymes that are involved in the degradation. In this work, two esterase-encoding genes (sulE, pnbA) and a glutathione-S-transferase-encoding gene (gst) responsible for the detoxification of chlorimuron-ethyl by strain Chenggangzhangella methanolivorans CHL1 were identified, then cloned and expressed in Escherichia coli BL21 (DE3). These key chlorimuron-ethyl-degrading enzymes are candidates for the construction of engineered bacteria to degrade this pesticide and enrich the resources for bioremediating environments polluted with sulfonylurea herbicides.

Induction of CD4+ regulatory T cells by stimulation with Staphylococcal Enterotoxin C2 through different signaling pathways.

As a member of superantigens, Staphylococcal Enterotoxin C2 (SEC2) can potently activate T cells expressing specific Vß repertoires and has been applied in clinic for tumor immunotherapy in China for more than 20 years. However, excessive activation of T cells by over-stimulation with superantigen are always followed by eliciting regulatory T cells (Tregs) induction and functional immunosuppression, which brings uncertainties to SEC2 application in tumor immunotherapy. In this study, we found that SEC2 could induce CD4+CD25+Foxp3+ Tregs from the murine splenocytes in dose and time related manners. The induced Tregs with high expression of GITR and CTLA-4 and low expression of CD127 were TCR Vß8.2-specific and have character of IL-10 production in a SEC2 dose-depended manner. Importantly, SEC2-induced CD4+ Tregs showed the potent capacity of suppressing proliferation of intact murine splenocytes response to SEC2. Furthermore, by using specific inhibitors or neutralizing antibody, we proved that the signaling pathways of TCR-NFAT/AP-1, IL-2-STAT5, and TGF-ß-Smad3 play crucial roles in Tregs induction by SEC2. These findings will help us better understand the balance of immune stimulation and immunosuppression mediated by SEC2 and provide valuable guidance for SEC2 application in antitumor immunology.

A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection.

Influenza pandemics pose public health threats annually for lacking vaccine that provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens that induce cross-protective antibody responses with epitopes that activate cross-protective T cell responses might be an attractive strategy for developing a universal vaccine. In this study, we constructed a recombinant protein named NMHC that consists of influenza viral conserved epitopes and a superantigen fragment. NMHC promoted the maturation of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2, and Th17 subtypes. Mice vaccinated with NMHC produced high levels of immunoglobulins that cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtype) and group 2 (H3 subtype) influenza virus strains. Furthermore, purified anti-NMHC antibodies bound to multiple HAs with high affinities. NMHC vaccination effectively protected mice from infection and lung damage when exposed to two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T cell responses that cleared the virus from infected tissues and prevented virus spread. In conclusion, this study provides proof of concept that NMHC vaccination triggers B and T cell immune responses against multiple influenza virus infections. Therefore, NMHC might be a candidate universal broad-spectrum vaccine for the prevention and treatment of multiple influenza viruses.

An Experiment Study on the Laminar Burning Velocity and Markstein Length of Chlorella Oil/RP-3 Kerosene Blends.

Experiments have been carried out in a constant volume chamber to investigate the effects of Chlorella oil addition on the laminar burning velocity and Markstein length of Chlorella oil/RP-3 kerosene blends at an initial pressure of 0.1 MPa and temperature of 450 K over a wide equivalence ratio range from 0.8 to 1.4. The result shows that at equivalence ratios of 0.9 and 1.1, with the increase of Chlorella oil addition, no cellular structure is observed in the flame propagation images. It means that the Chlorella oil addition has little effect on the flame stability under these experimental conditions; however, at an equivalence ratio of 1.3, with the increase of Chlorella oil addition from 0 to 0.5, the flame tends to be stable. It is found that the Markstein length of Chlorella oil/RP-3 blend decreases with the increase of the equivalence ratio. The blend with 0.5 Chlorella oil addition has a more rapid decrease in Markstein length compared with that of the RP-3 between the equivalence ratio from 1.1 to 1.3. The peak laminar burning velocity of Chlorella oil/RP-3 kerosene blend is obtained at the equivalence ratio of 1.1, and with the increase of Chlorella oil addition from 0 to 0.5, the laminar burning velocity increases about 20%.

An iRGD peptide fused superantigen mutant induced tumor-targeting and T lymphocyte infiltrating in cancer immunotherapy.

Solid tumors are intrinsically resistant to immunotherapy because of the major challenges including the immunosuppression and poor penetration of drugs and lymphocytes into solid tumors due to the complicated tumor microenvironment (TME). Our previous study has created a novel superantigen mutant ST-4 to efficiently active the T lymphocytes and alleviate immune suppression. In the present study, to accumulate ST-4 into the TME, we constructed a recombinant protein, ST-4-iRGD, by fusing ST-4 to a tumor-homing peptide, iRGD. We hypothesized that ST-4-iRGD could internalize into the TME through iRGD-mediated tumor targeting and tumor tissue penetrating to activate the regional immunoreaction. The results of in vitro studies showed that ST-4-iRGD achieved improved tumor targeting and cytotoxicity in mouse B16F10 melanoma cells. The iRGD-mediated tumor tissue penetration was further confirmed by imaging and immunofluorescence studies in vivo, wherein higher distribution of ST-4-iRGD was observed in the mouse 4T1 breast tumor model. Moreover, ST-4-iRGD exhibited enhanced anti-solid tumor characteristics and induced improved lymphocyte infiltration in the B16F10 and 4T1 models. In conclusion, using iRGD to facilitate better dissemination of the therapeutic agent ST-4 throughout a solid tumor mass is feasible, and ST-4-iRGD may be a potential candidate for efficient cancer immunotherapy in the future.

[Alpha-glycosidase helps the diagnosis of epididymal obstructive azoospermia].

OBJECTIVE: To assess the diagnostic value of alpha-glycosidase for epididymal lump induced obstructive azoospermia. METHODS: Seventy-six infertile men with normal spermatogenic function were divided according to sperm density into a normal density group (n = 27), an oligospermia group (n = 21) and an obstructive azoospermia group (n = 28), and another 30 fertile males were included as normal controls. Semen plasma alpha-glycosidase, leucocyte count, sexual hormone levels and the diameters of the epididymal lumps were measured and their correlations were analyzed. RESULTS: alpha-Glycosidase in the obstructive azoospermia group was significantly lower than that of the other groups (P <0.05), and negatively correlated with epididymal volume (r = -0.417, P <0.05) and leucocyte count (r = -0.342, P <0.05). CONCLUSION: Alpha-Glycosidase has been proved of diagnostic value for epididymal obstructive azoospermia.