Iron(I) Complex Bearing an Open-Shell Diazenido Ligand.

Low-valent transition-metal diazenido species are important intermediates in transition-metal-mediated dinitrogen reduction reactions. Isolable complexes of the type unanimously feature closed-shell diazenido ligands. Those bearing open-shell diazenido ligands have remained elusive. Herein, we report the synthesis, characterization, and reactivity of a d7 iron(I) complex featuring an open-shell silyldiazenido ligand, [(ICy)Fe(NNSiiPr3)(η2:η2-dvtms)] (1, ICy = 1,3-dicyclohexylimidazole-2-ylidene, dvtms = divinyltetramethyldisiloxane). Complex 1 is prepared in good yield by silylation of the iron(-I)-N2 complex [K(18-crown-6)][(ICy)Fe(N2)(η2:η2-dvtms)] with iPr3SiOTf and has been fully characterized by various spectroscopic methods. Theoretical studies, in combination with characterization data, established an S = 1/2 ground spin-state for 1 that can best be described as a quartet iron(I) center featuring an antiferromagnetically coupled triplet silyldiazenido ligand. The diazenido and alkene ligands in 1 are labile, as indicated by the facile disproportionation reaction of 1 at ambient temperature to transform into the iron(II) bis(diazenido) species [(ICy)(NNSiiPr3)2Fe(dvtms)Fe(NNSiiPr3)2(ICy)] (2) and the iron(0) species [(ICy)Fe(η2:η2-dvtms)] and also the alkene-exchange reaction of 1 with PhCH═CHBC8H14 to form [(ICy)Fe(NNSiiPr3)(η2-trans-PhCH═CHBC8H14)] (3). Complex 1 is light-sensitive. Upon photolysis, it undergoes a SiiPr3 radical-transfer reaction to yield [(ICy)Fe(σ:η2-MeCHSiMe2OSiMe2CH═CHSiiPr3)] (4) and N2. The reactions of 1 with the trityl radical and organic bromides yield iron(II) complexes, which indicates its reducing nature. Moreover, 1 is a weak hydrogen-atom abstractor, as indicated by its inertness toward HSi(SiMe3)3 and cyclohexa-1,4-diene and the low calculated N-H bond dissociation energy (48 kcal/mol) of its corresponding iron(II) iso-hydrazenido species.

Association of the live microbe intake from foods with all-cause and cardiovascular disease-specific mortality: a prospective cohort study.

BACKGROUND: Live dietary microbes have been hypothesized to promoting human health. However, there has been lacking perceptions to crystallize nexus between consumption of foods with live microbes and mortality. OBJECTIVE: To investigate the association of consumption of foods with medium to high amounts of live microbes with all-cause, cancer-specific, and cardiovascular disease (CVD)-specific mortality. METHODS: The data were obtained from the National Health and Nutrition Examination Survey 1999-2018 at baseline linked to the 2019 National Death Index records. Based on consumption of foods that were categorized as either having medium or high microbial content (MedHi foods), participants were classified into three groups. Kaplan-Meier survival curves and multivariable Cox regression models were used to estimate the association of consumption of MedHi foods with mortality. Population-attributable fractions (PAFs) of consumption of MedHi foods in relation to mortality risk were also estimated. RESULTS: A total of 35,299 adults aged ≥ 20 years were included in this study. During a median follow-up of 9.67 years, compared with adults in G1, those in G3 had 16% (hazard ratio [HR], 0.84; 95% confidence interval [CI], 0.77-0.90) reduced risk of all-cause mortality, and 23% (HR, 0.77; 95% CI, 0.67-0.89) reduced risk of CVD-specific mortality. The PAF of high (G3) vs. intermediate or low consumption of MedHi foods (G1 + G2) with all-cause and CVD-specific mortality was 3.4% and 4.3%, respectively. CONCLUSIONS: Consumption of foods with higher microbial concentrations is associated with a reduced risk of all-cause and CVD-specific mortality in US adults.

Performance evaluation of differential splicing analysis methods and splicing analytics platform construction.

A proportion of previously defined benign variants or variants of uncertain significance in humans, which are challenging to identify, may induce an abnormal splicing process. An increasing number of methods have been developed to predict splicing variants, but their performance has not been completely evaluated using independent benchmarks. Here, we manually sourced ∼50 000 positive/negative splicing variants from > 8000 studies and selected the independent splicing variants to evaluate the performance of prediction methods. These methods showed different performances in recognizing splicing variants in donor and acceptor regions, reminiscent of different weight coefficient applications to predict novel splicing variants. Of these methods, 66.67% exhibited higher specificities than sensitivities, suggesting that more moderate cut-off values are necessary to distinguish splicing variants. Moreover, the high correlation and consistent prediction ratio validated the feasibility of integration of the splicing prediction method in identifying splicing variants. We developed a splicing analytics platform called SPCards, which curates splicing variants from publications and predicts splicing scores of variants in genomes. SPCards also offers variant-level and gene-level annotation information, including allele frequency, non-synonymous prediction and comprehensive functional information. SPCards is suitable for high-throughput genetic identification of splicing variants, particularly those located in non-canonical splicing regions.

Induction of clastogenesis and gene mutations by carbamazepine (at its therapeutically effective serum levels) in mammalian cells and the dependence on human CYP2B6 enzyme activity.

Carbamazepine (CBZ, an antiepileptic) is metabolized by multiple CYP enzymes to its epoxide and hydroxides; however, whether it is genotoxic remains unclear. In this study, molecular docking (CBZ to CYPs) and cytogenotoxic toxicity assays were employed to investigate the activation of CBZ for mutagenic effects, in various mammalian cell models. Docking results indicated that CBZ was valid as a substrate of human CYP2B6 and 2E1, while not for CYP1A1, 1A2, 1B1 or 3A4. In the Chinese hamster (V79) cell line and its derivatives genetically engineered for the expression of human CYP1A1, 1A2, 1B1, 2E1 or 3A4 CBZ (2.5 ~ 40 µM) did not induce micronucleus, while in human CYP2B6-expressing cells CBZ significantly induced micronucleus formation. In a human hepatoma C3A cell line, which endogenously expressed CYP2B6 twofold higher than in HepG2 cells, CBZ induced micronucleus potently, which was blocked by 1-aminobenzotriazole (inhibitor of CYPs) and ticlopidine (specific CYP2B6 inhibitor). In HepG2 cells CBZ did not induce micronucleus; however, pretreatment of the cells with CICTO (CYP2B6 inducer) led to micronucleus formation by CBZ, while rifampicin (CYP3A4 inducer) or PCB126 (CYP1A inducer) did not change the negative results. Immunofluorescent assay showed that CBZ selectively induced centromere-free micronucleus. Moreover, CBZ induced double-strand DNA breaks (γ-H2AX elevation, by Western blot) and PIG-A gene mutations (by flowcytometry) in C3A (threshold being 5 µM, lower than its therapeutic serum concentrations, 17 ~ 51 µM), with no effects in HepG2 cells. Clearly, CBZ may induce clastogenesis and gene mutations at its therapeutic concentrations, human CYP2B6 being a major activating enzyme.

A Type Ib Crustin from Deep-Sea Shrimp Possesses Antimicrobial and Immunomodulatory Activity.

Crustins are small antimicrobial proteins produced by crustaceans. Of the many reported crustins, very few are from deep sea environments. Crustins are categorized into several types. Recently, the Type I crustin has been further classified into three subtypes, one of which is Type Ib, whose function is unknown. Here, we studied the function of a Type Ib crustin (designated Crus2) identified from a deep-sea crustacean. Crus2 has a whey acidic protein (WAP) domain and a long C-terminal region (named P58). Recombinant Crus2 bound to peptidoglycan (PGN), lipoteichoic acid (LTA), and lipopolysaccharide (LPS), and killed Gram-positive and Gram-negative bacteria by permeabilizing the bacterial cytomembrane. Consistently, Crus2 dramatically attenuated the inflammatory response induced by LPS and LTA. Disruption of the disulfide bonds in the WAP domain abolished the bactericidal ability of Crus2, but had no effect on the bacterial binding ability of Crus2. Deletion of the C-terminal P58 region moderately affected the antimicrobial activity of Crus2 against some bacteria. P58 as a synthesized peptide could bind bacteria and inhibit the bactericidal activity of Crus2. Taken together, these results revealed different roles played by the WAP domain and the P58 region in Type Ib crustin, and provided new insights into the antimicrobial and immunomodulatory functions of crustins.

Induction and Monitoring of DNA Phase Separation in Living Cells by a Light-Switching Ruthenium Complex.

Phase separation of DNA is involved in chromatin packing for the regulation of gene transcription. Visualization and manipulation of DNA phase separation in living cells present great challenges. Herein, we present a Ru(II) complex (Ru1) with high DNA binding affinity and DNA "light-switch" behavior that can induce and monitor DNA phase separation both in vitro and in living cells. Molecular dynamics simulations indicate that the two phen-PPh3 ligands with positively charged lipophilic triphenylphosphine substituents and flexible long alkyl chains in Ru1 play essential roles in the formation of multivalent binding forces between DNA molecules to induce DNA phase separation. Importantly, the unique environmental sensitive emission property of Ru1 enables direct visualization of the dynamic process of DNA phase separation in living cells by two-photon phosphorescent lifetime imaging. Moreover, Ru1 can change the gene expression pattern by modulating chromatin accessibility as demonstrated by integrating RNA-sequencing and transposase-accessible chromatin with high-throughput sequencing. In all, we present here the first small-molecule-based tracer and modulator of DNA phase separation in living cells and elucidate its impact on the chromatin state and transcriptome.

Influence of Bisphenol Compounds at Nanomolar Concentrations on Chromosome Damage Induced by Metabolically Activated Carcinogens in HepG2 Cells.

Bisphenol (BP) compounds are endocrine-disrupting organic pollutants. BPs may increase the messenger RNA (mRNA) transcripts of nuclear receptors (NRs) regulating the expression of xenobiotic-metabolizing cytochrome P450 (CYP) enzymes. Their impact on the genotoxicity of metabolically activated carcinogens, however, remains unknown. In this study, effects of the bisphenols A, F, S, and AF on the expression of the aryl hydrocarbon receptor (AhR), the pregnane X receptor (PXR), the constitutive androstane receptor, and individual xenobiotic-metabolizing CYP enzymes in a human hepatoma (HepG2) cell line were investigated, along with in silico binding studies of BPs to each receptor. The results indicated that each BP at 1 to 100 nM concentrations increased the mRNA transcripts and protein levels of AhR, PXR, CYP1A1, 1A2, 1B1, 2E1, and 3A4. The predicted affinities of the BPs for binding AhR were comparable to those of potent agonists. Pretreatment of HepG2 cells with each BP potentiated the induction of micronuclei by benzo[a]pyrene, aflatoxin B1, benzene, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; this effect was abolished/reduced by inhibitors of NRs and/or CYPs. Our study suggests that BPs at human exposure levels may aggravate chromosome damage by several impactful carcinogens in human cells by inducing relevant CYP enzymes, mostly via NR modulation.

A Crustin from Hydrothermal Vent Shrimp: Antimicrobial Activity and Mechanism.

Crustin is a type of antimicrobial peptide and plays an important role in the innate immunity of arthropods. We report here the identification and characterization of a crustin (named Crus1) from the shrimp Rimicaris sp. inhabiting the deep-sea hydrothermal vent in Manus Basin (Papua New Guinea). Crus1 shares the highest identity (51.76%) with a Type I crustin of Penaeus vannamei and possesses a whey acidic protein (WAP) domain, which contains eight cysteine residues that form the conserved 'four-disulfide core' structure. Recombinant Crus1 (rCrus1) bound to peptidoglycan and lipoteichoic acid, and effectively killed Gram-positive bacteria in a manner that was dependent on pH, temperature, and disulfide linkage. rCrus1 induced membrane leakage and structure damage in the target bacteria, but had no effect on bacterial protoplasts. Serine substitution of each of the 8 Cys residues in the WAP domain did not affect the bacterial binding capacity but completely abolished the bactericidal activity of rCrus1. These results provide new insights into the characteristic and mechanism of the antimicrobial activity of deep sea crustins.

Radiomics nomogram for differentiating between benign and malignant soft-tissue masses of the extremities.

BACKGROUND: Preoperative differentiation between malignant and benign tumors is important for treatment decisions. PURPOSE/HYPOTHESIS: To investigate/validate a radiomics nomogram for preoperative differentiation between malignant and benign masses. STUDY TYPE: Retrospective. POPULATION: Imaging data of 91 patients. FIELD STRENGTH/SEQUENCE: T1 -weighted images (570 msec repetition time [TR]; 17.9 msec echo time [TE], 200-400 mm field of view [FOV], 208-512 × 208-512 matrix), fat-suppressed fast-spin-echo (FSE) T2 -weighted images (T2 WIs) (4331 msec TR; 87.9 msec TE, 200-400 mm FOV, 312 × 312 matrix), slice thickness 4 mm, and slice spacing 1 mm. ASSESSMENT: Fat-suppressed FSE T2 WIs were selected for extraction of features. Radiomics features were extracted from fat-suppressed T2 WIs. A radiomics signature was generated from the training dataset using least absolute shrinkage and selection operator algorithms. Independent risk factors were identified by multivariate logistic regression analysis and a radiomics nomogram was constructed. Nomogram capability was evaluated in the training dataset and validated in the validation dataset. Performance of the nomogram, radiomics signature, and clinical model were compared. STATISTICAL TESTS: 1) Independent t-test or Mann-Whitney U-test: for continuous variables. Fisher's exact test or χ2 test: comparing categorical variables between two groups. Univariate analysis: evaluating associations between clinical/morphological characteristics and malignancy. 2) Least absolute shrinkage and selection operator (LASSO)-logistic regression model: selection of malignancy features. 3) Significant clinical/morphological characteristics and radiomics signature were input variables for multiple logistic regression analysis. Area under the curve (AUC): evaluation of ability of the nomogram to identify malignancy. Hosmer-Lemeshow test and decision curve: evaluation and validation of nomogram results. RESULTS: The radiomics nomogram was able to differentiate malignancy from benignity in the training and validation datasets with an AUC of 0.94. The nomogram outperformed both the radiomics signature and clinical model alone. DATA CONCLUSION: This radiomics nomogram is a noninvasive, low-cost preoperative prediction method combining the radiomics signature and clinical model. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:155-163.

Y75B8A.8 (HC8) protein of Haemonchus contortus: A functional inhibitor of host IL-2.

Interleukin 2 (IL-2) is an important immune regulatory factor in the immune response of the host. However, little is known about the inhibitor of host IL-2 in Haemonchus contortus infection. In this study, we found that globin domain-containing protein (HCGB) and Protein Y75B8A.8 (HC8) from H contortus excretory and secretory products are two binding proteins of IL-2 in goats. The yeast two-hybrid screening further validated the positive interactions of IL-2 with HCGB and HC8. Meanwhile, we found that HC8 had inhibitory effects on IL-2-induced peripheral blood mononuclear cell (PBMC) proliferation, while HCGB did not. Furthermore, transcriptional analysis revealed that HC8 could block the IL-2-activated signalling pathway. Our results showed that HC8 was a functional inhibitor of goat IL-2.

Molecular dynamics investigation of the local structure in iron melts and its role in crystal nucleation during rapid solidification.

A comprehensive investigation on local structures in iron melts and their role in nucleation under various cooling rates was performed by means of large-scale molecular dynamics simulations. The embedded atoms method (EAM) was adopted to describe the interactions between iron atoms. Connections between short-range order (SRO), medium-range order (MRO), and crystalline nucleation from iron melts were constructed using several structural analysis techniques, including the radial distribution function, common neighbor analysis method, the Voronoi tessellation, and bond order analysis. The simulation results showed that abundant types of atomic clusters with SRO, mainly including the icosahedral-like (ICO-like) and fcc-like clusters, were predominant in undercooled iron melts. The obtained microstructures were determined by the competition between the ICO-like and crystal-like configurations. There existed a critical cooling rate, below which the fcc-like configurations gain the advantage upon cooling and where crystallization could take place; otherwise, the ICO-like configurations are favored and the glass phases could be obtained. Furthermore, it was proved that the crystal nucleation could be divided into three stages: first, a fluctuation and competition between crystal-like and ICO-like clusters in undercooled melts; second, the formation and growth of MRO clusters via the transformation of atomic configurations from ICO-like to crystal-like; finally, the nucleation of bcc nuclei from the core of steady MRO clusters. This process agrees with the Ostwald's step rule and the findings from other investigations. Based on the analysis of the compositional origin of MRO clusters, we further found that the MRO clusters were mainly composed of fcc-like instead of ICO-like configurations, indicating a negative role of ICO-like configurations in crystal nucleation.

Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

Recombinant protein of Haemonchus contortus 14-3-3 isoform 2 (rHcftt-2) decreased the production of IL-4 and suppressed the proliferation of goat PBMCs in vitro.

14-3-3 proteins have been found to be an excreted/secreted antigen and assumed to be released into the host-parasite interface and described in several unicellular and multicellular parasites. However, little is known about the immunomodulatory effects of H. controtus 14-3-3 protein on host cell. In present study, 14-3-3 isoform 2 gene, designated as Hcftt-2, was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the adult H. contortus cDNA and cloned into expression plasmid pET32a (+) and expression of the recombinant protein (rHcftt-2) was induced by IPTG. Binding activity of rHcftt-2 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and modulatory effects on cytokine production, cell proliferation, cell migration and nitric oxide (NO) production were observed by co-incubation of rHcftt-2 with goat PBMCs. Sequence analysis showed that it had significant homology with the known 14-3-3 protein isoform 2. Results of IFA revealed that, the rHcftt-2 was bound to the cell surface. We found that, the productions of IL10, IL-17, IFN-γ and cell migration of PBMCs were increased after the cells were incubated with rHCftt-2. However, the productions of IL-4, NO and cell proliferation of the PBMCs were significantly decreased in dose depended manner. Our results showed that the Hcftt-2 played important suppressive regulatory effects on the goat PBMCs.

Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii elongation factor 1-alpha.

BACKGROUND: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The high incidence and severe damage that can be caused by T. gondii infection clearly indicates the need for the development of a vaccine. T. gondii elongation factor 1-alpha (TgEF-1α) plays an important role in pathogenesis and host cell invasion for this parasite. The aim of this study was to evaluate the immune protective efficacy of a DNA vaccine encoding TgEF-1α gene against acute T. gondii infection in mice. METHODS: A DNA vaccine (pVAX-EF-1α) encoding T. gondii EF-1a (TgEF-1α) gene was constructed and its immune response and protective efficacy against lethal challenge in BALB/c mice were evaluated. RESULTS: Mice inoculated with the pVAX-EF-1α vaccine had a high level of specific anti-T. gondii antibodies and produced high levels of IFN-gamma, interleukin (IL)-4, and IL-17. The expression levels of MHC-I and MHC-II molecules as well as the percentages of both CD4(+) and CD8(+) T cells in mice vaccinated with pVAX-EF-1α were significantly increased (p < 0.05), compared with those in all the mice from control groups (blank control, PBS, and pVAXI). Immunization with pVAX-EF-1α significantly (p < 0.05) prolonged mouse survival time to 14.1 ± 1.7 days after challenge infection with the virulent T. gondii RH strain, compared with mice in the control groups which died within 8 days. CONCLUSIONS: DNA vaccination with pVAX-EF-1α triggered strong humoral and cellular responses and induced effective protection in mice against acute T. gondii infection, indicating that TgEF-1α is a promising vaccine candidate against acute toxoplasmosis.

Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides.

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773 bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13 kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-2 genes' expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.

RSSI-based smooth localization for indoor environment.

Radio frequency (RF) technique, for its better penetrability over traditional techniques such as infrared or ultrasound, is widely used for indoor localization and tracking. In this paper, three novel measurements, point decision accuracy, path matching error and wrong jumping ratio, are firstly defined to express the localization efficiency. Then, a novel RSSI-based smooth localization (RSL) algorithm is designed, implemented, and evaluated on the WiFi networks. The tree-based mechanism determines the current position and track of the entity by assigning the weights and accumulative weights for all collected RSSI information of reference points so as to make the localization smooth. The evaluation results indicate that the proposed algorithm brings better localization smoothness of reducing 10% path matching error and 30% wrong jumping ratio over the RADAR system.

[A long-term follow-up of the correlation between obstructive sleep apnea hypopnea syndrome and multiple organ diseases].

OBJECTIVE: To explore the correlation between obstructive sleep apnea hypopnea syndrome (OSAHS) and multiple organ diseases. METHODS: Home-visit questionnaires were performed in 1 868 subjects (956 male, 912 female) with an average age of (79 ± 5) years, and a prospective follow-up was performed for a period of 20 years with annual medical examinations. Multiple organ diseases included hypertension, coronary heart disease, stroke, diabetes, pulmonary heart disease, renal insufficiency and erythrocytosis. The subjects were grouped by the diagnosis of OSAHS. RESULTS: Among the 1 868 subjects, 598 (32.0%) were diagnosed with OSAHS, and 1 270 (68.0%) of non-OSAHS as the control group. By the end of follow-up, in the OSAHS group there were 477 (79.8%) cases with hypertension, 337 (56.4%) cases with coronary heart disease, 167 (27.9%) cases with stroke, 76 (12.7%) cases with diabetes, 37 (6.2%) cases with pulmonary heart disease, 73 (12.2%) cases with renal insufficiency and 211(35.3%) cases with erythrocytosis, all of which were significantly higher than those of the control group [323(25.4%), 315 (24.8%), 95 (7.5%), 69 (5.4%), 40 (3.2%), 58 (4.6%), 30 (2.4%), P < 0.01]. The number of diseases in the OSAHS group was also higher than that in the control group (P < 0.05) . CONCLUSION: The incidence of multiple organ diseases was remarkably higher in the OSAHS group than that in the control group, which indicated that OSAHS was a risk factor for multiple organ diseases. These diseases in OSAHS patients may be related to hypoxia caused by OSAHS, endocrine and metabolic disorders, unhealthy lifestyles and arteriosclerosis.

Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner.

Bacillus cereus is a clinically significant foodborne pathogen that causes severe gastrointestinal and non-gastrointestinal disease. Cereolysin O (CLO) is a putative virulence factor of B. cereus, and its function remains to be investigated. In this study, we examined the biological activity of CLO from a deep sea B. cereus isolate. CLO was highly toxic to mammalian cells and triggered pyroptosis through NLRP3 inflammasome-mediated caspase 1 and gasdermin D activation. CLO-induced cell death involved ROS accumulation and K+ efflux, and was blocked by serum lipids. CLO bound specifically to cholesterol, and this binding was essential to CLO cytotoxicity. The structural integrity of the three tryptophan residues in the C-terminal undecapeptide was vital for CLO to interact with membrane lipids and cause membrane perforation. Taken together, these results provided new insights into the molecular mechanism of B. cereus CLO-mediated cytotoxicity.

Haemolysins are essential to the pathogenicity of deep-sea Vibrio fluvialis.

Vibrio fluvialis is an emerging foodborne pathogen that produces VFH (Vibrio fluvialis hemolysin) and δVFH (delta-Vibrio fluvialis hemolysin). The function of δVFH is unclear. Currently, no pathogenic V. fluvialis from deep sea has been reported. In this work, a deep-sea V. fluvialis isolate (V13) was examined for pathogenicity. V13 was most closely related to V. fluvialis ATCC 33809, a human isolate, but possessed 262 unique genes. V13 caused lethal infection in fish and induced pyroptosis involving activation of the NLRP3 inflammasome, caspase 1 (Casp1), and gasdermin D (GSDMD). V13 defective in VFH or VFH plus δVFH exhibited significantly weakened cytotoxicity. Recombinant δVFH induced NLRP3-Casp1-GSDMD-mediated pyroptosis in a manner that depended on K+ efflux and intracellular Ca2+ accumulation. δVFH bound several plasma membrane lipids, and these bindings were crucial for δVFH cytotoxicity. Together these results provided new insights into the function of δVFH and the virulence mechanism of V. fluvialis.

Infection status and molecular detection of pathogens carried by ectoparasites of Miniopterus fuliginosus bats in Yunnan, China.

Bats serve as natural hosts for various infectious agents that can affect both humans and animals, and they are geographically widespread. In recent years, the prevalence of bat-associated pathogens has surged on a global scale, consequently generating significant interest in bats and their ectoparasites. In this study, we specifically selected the Miniopterus fuliginosus as the host and conducted bat captures in Nanjian Yi Autonomous County, Dali Bai Autonomous Prefecture, and the other in Mouding Township, Chuxiong Yi Autonomous Prefecture, located in Yunnan Province, China. Ectoparasites were meticulously collected from the bat body surface, alongside blood samples for subsequent analyses. Following collection, the ectoparasites were methodically identified and subjected to comprehensive ecological analysis. Additionally, DNA was extracted from both the bat blood and bat flies, with conventional PCR techniques utilized for molecular screening of four pathogens: Anaplasma sp., Babesia sp., Hepatozoon sp., and Bartonella sp. The capture efforts yielded a total of 37 M. fuliginosus, from which 388 ectoparasites were recovered, including 197 gamasid mites (Cr = 50.77%, PM = 94.59%, MA = 5.32, MI = 5.63) and 191 bat flies (Cr = 49.23%, PM = 75.68%, MA = 5.16, MI = 6.82). Notably, Steatonyssus nyctali (Y = 0.28, m*/m = 2.44) and Nycteribia allotopa (Y = 0.23,m*/m = 1.54) predominated among different individuals of M. fuliginosus, exhibiting an aggregated distribution pattern. The infection rates of Bartonella sp. were identified to be 18.92% (7/37) among bats and 37.17% (71/191) among bat flies, based on the testing of 37 bats and 191 bat flies. Phylogenetic analysis demonstrated that the Bartonella sequences exhibited similarity to those found in bats and bat flies within China and South Korea. This study not only contributes to our comprehension of ectoparasite infection in M. fuliginosus but also establishes a foundation for potential exploration of their role as vectors.