Molecular basis of TMPRSS2 recognition by Paeniclostridium sordellii hemorrhagic toxin.

Hemorrhagic toxin (TcsH) is a major virulence factor produced by Paeniclostridium sordellii, which is a non-negligible threat to women undergoing childbirth or abortions. Recently, Transmembrane Serine Protease 2 (TMPRSS2) was identified as a host receptor of TcsH. Here, we show the cryo-EM structures of the TcsH-TMPRSS2 complex and uncover that TcsH binds to the serine protease domain (SPD) of TMPRSS2 through the CROP unit-VI. This receptor binding mode is unique among LCTs. Five top surface loops of TMPRSS2SPD, which also determine the protease substrate specificity, constitute the structural determinants recognized by TcsH. The binding of TcsH inhibits the proteolytic activity of TMPRSS2, whereas its implication in disease manifestations remains unclear. We further show that mutations selectively disrupting TMPRSS2-binding reduce TcsH toxicity in the intestinal epithelium of the female mice. These findings together shed light on the distinct molecular basis of TcsH-TMPRSS2 interactions, which expands our knowledge of host recognition mechanisms employed by LCTs and provides novel targets for developing therapeutics against P. sordellii infections.

Topological links in predicted protein complex structures reveal limitations of AlphaFold.

AlphaFold is making great progress in protein structure prediction, not only for single-chain proteins but also for multi-chain protein complexes. When using AlphaFold-Multimer to predict protein‒protein complexes, we observed some unusual structures in which chains are looped around each other to form topologically intertwining links at the interface. Based on physical principles, such topological links should generally not exist in native protein complex structures unless covalent modifications of residues are involved. Although it is well known and has been well studied that protein structures may have topologically complex shapes such as knots and links, existing methods are hampered by the chain closure problem and show poor performance in identifying topologically linked structures in protein‒protein complexes. Therefore, we address the chain closure problem by using sliding windows from a local perspective and propose an algorithm to measure the topological-geometric features that can be used to identify topologically linked structures. An application of the method to AlphaFold-Multimer-predicted protein complex structures finds that approximately 1.72% of the predicted structures contain topological links. The method presented in this work will facilitate the computational study of protein‒protein interactions and help further improve the structural prediction of multi-chain protein complexes.

Accumulation, translocation, and fractionation of rare earth elements (REEs) in fern species of hyperaccumulators and non-hyperaccumulators growing in urban areas.

The issue of ion-adsorption type rare earth deposits (IADs) in urban areas of South China has garnered significant attention due to its environmental implications. Hyperaccumulator-based phytoremediation is a potentially effective solution for reducing the environmental impact of IADs in urban areas, particularly using ferns as they are known to be REE hyperaccumulators. However, the ability of different fern species to accumulate REEs in urban areas remains unknown. In this study, four fern species, including known hyperaccumulators (Dicranopteris linearis and Blechnum orientale) and other ferns (Pteris ensiformis and Cibotium barometz), were studied to investigate their REE accumulation abilities in the Guangzhou urban area. The aboveground parts of Dicranopteris linearis (848.7 µg g-1) and Blechum orientale (1046.8 µg g-1) have been found to accumulate high concentrations of REEs, demonstrating they probably can be applied for phytoremediation in the natural environments. Despite having lower REE concentrations than REE hyperaccumulators, Pteris ensiformis and Cibotium barometz still probably have the function as phytostabilizers in urban areas, as REEs can be enriched in their roots beyond the normal levels of plants. The enrichment of REEs in ferns is influenced by the availability of various nutrients (K, Ca, Fe, and P), which probably can be associated with different growth processes. The four fern species show LREE enrichment, moderate Eu anomalies and different Ce anomalies. It is difficult to absorb and transfer Ce to the aboveground parts of Blechnum orientale and Cibotium barometz. The study also identified selective enrichment of Ce in Pteris ensiformis, which has potential for comprehensive extraction of REEs when combined with other REE hyperaccumulators. REE fractionations are probably determined by the specific characteristics of different fern parts. Overall, these findings provide insights for addressing potential environmental problems related to IADs and offer guidelines for phytoremediation technology in addressing high REE levels in urban areas.

LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin.

Clostridium novyi alpha-toxin (Tcnα) is a potent exotoxin that induces severe symptoms including gas gangrene, myositis, necrotic hepatitis, and sepsis. Tcnα binds to sulfated glycosaminoglycans (sGAG) for cell-surface attachment and utilizes low-density lipoprotein receptor (LDLR) for rapid entry. However, it was also shown that Tcnα may use alternative entry receptors other than LDLR. Here, we define that LRP1 and Megalin can also facilitate the cellular entry of Tcnα by employing reconstitutive LDLR family proteins. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (also known as LDL receptor type A repeats). Notably, LDLR and LRP1 have contrasting expression levels in many different cells, thus the dominant entry receptor for Tcnα could be cell-type dependent. These findings together increase our knowledge of the Tcnα actions and further help to understand the pathogenesis of C. novyi infection-associated diseases.

Protocol for genome-wide CRISPR knockout screens of bacterial cytotoxins in HeLa cells.

CRISPR screening is a powerful tool to identify host factors for pathogenic agents including viruses and bacterial toxins. Here, we present a protocol to conduct a genome-scale CRISPR screen on HeLa cells for host factors involved in the toxin action of Clostridioides difficile TcdB4. We describe in detail how to prepare the library, set up the screen, obtain the gene sequences, and analyze the results. This protocol can also be modified for other genome-scale libraries, cell lines, and cytotoxins. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).

Paeniclostridium sordellii hemorrhagic toxin targets TMPRSS2 to induce colonic epithelial lesions.

Hemorrhagic toxin (TcsH) is an important exotoxin produced by Paeniclostridium sordellii, but the exact role of TcsH in the pathogenesis remains unclear, partly due to the lack of knowledge of host receptor(s). Here, we carried out two genome-wide CRISPR/Cas9 screens parallelly with TcsH and identified cell surface fucosylation and TMPRSS2 as host factors contributing to the binding and entry of TcsH. Genetic deletion of either fucosylation biosynthesis enzymes or TMPRSS2 in the cells confers resistance to TcsH intoxication. Interestingly, TMPRSS2 and fucosylated glycans can mediate the binding/entry of TcsH independently, thus serving as redundant receptors. Both TMPRSS2 and fucosylation recognize TcsH through its CROPs domain. By using Tmprss2‒/‒ mice, we show that Tmprss2 is important for TcsH-induced systematic toxicity and colonic epithelial lesions. These findings reveal the importance of TMPRSS2 and surface fucosylation in TcsH actions and further provide insights into host recognition mechanisms for large clostridial toxins.

Nickel sulfide-oxide heterostructured electrocatalysts: Bi-functionality for overall water splitting and in-situ reconstruction.

Bi-functional electrocatalysts are desired for both hydrogen and oxygen evolution reactions (HER and OER). Herein, facile O2-plasma activation is introduced to improve the bi-functionality via constructing nickel sulfide-oxide heterostructures. Ni3S2-NiOx supported by nickel foam delivers obviously elevated HER and OER activity in comparison with pristine Ni3S2 and recently reported NiSx-based electrocatalysts, featured by the low overpotentials for HER (104 mV) and OER (241 mV) at ±10 mA cm-2 in 1.0 M KOH, as well as a voltage of 1.52 V for overall water splitting. As revealed by in-situ Raman spectroscopy, on the one hand, Ni(OH)2 generated from Ni3S2 during alkaline HER accelerates water dissociation toward the gradually improved performance; on the other hand, this heterostructure undergoes extensive oxidation during OER, leading to excessive NiOOH covering on Ni3S2 and thereby declining activity. These changes are interpreted by the distinct thermodynamic relationship under specific electrochemical conditions via density functional theory calculations.

An Atypical Case of Monomicrobial Clostridioides difficile Septicemia With No Gastrointestinal Manifestations.

An uncommon case of monomicrobial Clostridioides difficile septicemia in a 63-year-old man was reported in Zhejiang, China. Once diagnosed, vancomycin treatment cleared the infections. The patient had no remarkable medical history, and the inspection showed no overt gastrointestinal symptoms, though C. difficile was detected in his stool samples. However, we later defined that the C. difficile strain isolated from the blood sample was different from the one isolated from his stool using the whole genome sequencing analysis. By retrospective analysis of his medical record, we noticed that the man had a recent tooth extraction thus the bacterium may have invaded through the root canal. Therefore, we suggest that oral C. difficile colonization may be a potential risk factor for severe C. difficile septicemia, which could be clinically alarming.

Distinctive signatures of pathogenic and antibiotic resistant potentials in the hadal microbiome.

BACKGROUND: Hadal zone of the deep-sea trenches accommodates microbial life under extreme energy limitations and environmental conditions, such as low temperature, high pressure, and low organic matter down to 11,000 m below sea level. However, microbial pathogenicity, resistance, and adaptation therein remain unknown. Here we used culture-independent metagenomic approaches to explore the virulence and antibiotic resistance in the hadal microbiota of the Mariana Trench. RESULTS: The results indicate that the 10,898 m Challenger Deep bottom sediment harbored prosperous microbiota with contrasting signatures of virulence factors and antibiotic resistance, compared with the neighboring but shallower 6038 m steep wall site and the more nearshore 5856 m Pacific basin site. Virulence genes including several famous large translocating virulence genes (e.g., botulinum neurotoxins, tetanus neurotoxin, and Clostridium difficile toxins) were uniquely detected in the trench bottom. However, the shallower and more nearshore site sediment had a higher abundance and richer diversity of known antibiotic resistance genes (ARGs), especially for those clinically relevant ones (e.g., fosX, sul1, and TEM-family extended-spectrum beta-lactamases), revealing resistance selection under anthropogenic stresses. Further analysis of mobilome (i.e., the collection of mobile genetic elements, MGEs) suggests horizontal gene transfer mediated by phage and integrase as the major mechanism for the evolution of Mariana Trench sediment bacteria. Notably, contig-level co-occurring and taxonomic analysis shows emerging evidence for substantial co-selection of virulence genes and ARGs in taxonomically diverse bacteria in the hadal sediment, especially for the Challenger Deep bottom where mobilized ARGs and virulence genes are favorably enriched in largely unexplored bacteria. CONCLUSIONS: This study reports the landscape of virulence factors, antibiotic resistome, and mobilome in the sediment and seawater microbiota residing hadal environment of the deepest ocean bottom on earth. Our work unravels the contrasting and unique features of virulence genes, ARGs, and MGEs in the Mariana Trench bottom, providing new insights into the eco-environmental and biological processes underlying microbial pathogenicity, resistance, and adaptative evolution in the hadal environment.

TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

Visible light-driven efficient palladium catalyst turnover in oxidative transformations within confined frameworks.

Palladium catalyst turnover by reoxidation of a low-valent Pd species dominates the proceeding of an efficient oxidative transformation, but the state-of-the-art catalysis approaches still have great challenges from the perspectives of high efficiency, atom-economy and environmental-friendliness. Herein, we report a new strategy for addressing Pd reoxidation problem by the fabrication of spatially proximate IrIII photocatalyst and PdII catalyst into metal-organic framework (MOF), affording MOFs based Pd/photoredox catalysts UiO-67-Ir-PdX2 (X = OAc, TFA), which are systematically evaluated using three representative Pd-catalyzed oxidation reactions. Owing to the stabilization of single-site Pd and Ir catalysts by MOFs framework as well as the proximity of them favoring fast electron transfer, UiO-67-Ir-PdX2, under visible light, exhibits up to 25 times of Pd catalyst turnover number than the existing catalysis systems. Mechanism investigations theoretically corroborate the capability of MOFs based Pd/photoredox catalysis to regulate the competitive processes of Pd0 aggregation and reoxidation in Pd-catalyzed oxidation reactions.

Lamin-A interacting protein Hsp90 is required for DNA damage repair and chemoresistance of ovarian cancer cells.

Ovarian cancer is the most malignant gynecologic cancer. Previous studies found that lamin-A was associated with DNA damage repair proteins but the underlying mechanism remains unclear. We speculate that this may be related to its interacting proteins, such as Hsp90. The aim of this study is to investigate the effects of Hsp90 on DNA damage repair and chemoresistance of ovarian cancer cells. In our research, co-immunoprecipitation (co-IP) and mass spectrometry (MS) were used to identify proteins interacting with lamin-A and the interaction domain. Next, the relationship between lamin-A and Hsp90 was explored by Western blotting (WB) and immunofluorescence staining. Then, effect of Hsp90 inhibition on DNA damage repair was assessed through detecting Rad50 and Ku80 by WB. Furthermore, to test the roles of 17-AAG on cell chemosensitivity, CCK-8 and colony formation assay were carried out. Meanwhile, IC50 of cells were calculated, followed by immunofluorescence to detect DNA damage. At last, the mouse xenograft model was used in determining the capacity of 17-AAG and DDP to suppress tumor growth and metastatic potential. The results showed that lamin-A could interact with Hsp90 via the domain of lamin-A1-430. Besides, the distribution of Hsp90 could be affected by lamin-A. After lamin-A knockdown, Hsp90 decreased in the cytoplasm and increased in the nucleus, suggesting that the interaction between lamin-A and Hsp90 may be related to the nucleocytoplasmic transport of Hsp90. Moreover, inhibition of Hsp90 led to an obvious decrease in the expression of DSBs (DNA double-strand break) repair proteins, as well as cell proliferation ability upon DDP treatment and IC50 of DDP, causing more serious DNA damage. In addition, the combination of 17-AAG and DDP restrained the growth of ovarian cancer efficiently in vivo and prolonged the survival time of tumor-bearing mice.

Extraction of alumina from aluminum dross by a non-hazardous alkaline sintering process: Dissolution kinetics of alumina and silica from calcined materials.

The aluminum dross (AD), which causes numerous problems of its management and disposal to environment is a useful resource to extract alumina. This study explains a novel process to extract highly pure alumina (Al2O3) from AD at a high extraction rate without producing the residues and exhaust gases. An experimental set up was designed to perform the grinding of AD for the decomposition of aluminum nitride (AlN) and the removal of salts. Thereby, the desalted dross was used to detect the optimum alkaline (NaOH) calcination parameters and leaching conditions, as well as the dissolution kinetics of alumina and silica. The leaching residues were used to produce Ettringite mineral with calcium-based compounds (including CaO and CaSO4) to avoid the problems of solid waste disposal from the leaching process. Moreover, to purify the alumina, slightly soluble CaSO4 was added in leaching solution to precipitate silicate and the optimum additive/solution ratio (g/mL) was determined. The aluminum hydroxide (Al(OH)3), precipitated after the carbonization was calcinated at 900.0 °C for 2 h to produce γ-alumina. The morphological and mineralogical characterizations of AD, γ-Al2O3 and the synthesized Ettringite mineral were studied by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and X-ray fluorescence (XRF). It was observed that activation temperature of 1000.0 °C, Na2O/Al2O3 molar ratio of 1.4, leaching temperature of 60.0 °C, leaching time of 40.0 min, and the leaching liquid/solid ratio (mL/g) of 25/1 were the optimal parameter conditions to extract alumina with the extraction rate at 86.7% and purity of more than 98%. The results of leaching kinetics' study showed that the dissolution of alumina and silica were both controlled by layer diffusion process with the apparent activation energy of 11.4010 kJ·mol-1 and 2.0556 kJ·mol-1, respectively.

Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology.

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B.

Clostridioides difficile toxins (TcdA and TcdB) are major exotoxins responsible for C. difficile infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269 C. difficile genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the tcdB gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of C. difficile. These findings suggest that the tcdB genes not only frequently mutate, but also continuously transfer and exchange among C. difficile strains.

Molybdenum Carbide-Oxide Heterostructures: In Situ Surface Reconfiguration toward Efficient Electrocatalytic Hydrogen Evolution.

Heterostructured Mo2 C-MoOx on carbon cloth (Mo2 C-MoOx /CC), as a model of easily oxidized electrocatalysts under ambient conditions, is investigated to uncover surface reconfiguration during the hydrogen evolution reaction (HER). Raman spectroscopy combined with electrochemical tests demonstrates that the MoVI oxides on the surface are in situ reduced to MoIV , accomplishing promoted HER in acidic condition. As indicated by density functional theoretical calculations, the in situ reduced surface with terminal Mo=O moieties can effectively bring the negative ΔGH* on bare Mo2 C close to a thermodynamic neutral value, addressing difficult H* desorption toward fast HER kinetics. The optimized Mo2 C-MoOx /CC only requires a low overpotential (η10 ) of 60 mV at -10 mA cm-2 in 1.0 m HClO4 , outperforming Mo2 C/CC and most non-precious electrocatalysts. In situ surface reconfiguration are shown on W2 C-WOx , highlighting the significance to boost various metal-carbides and to identify active sites.

Ovarian cancer cell-secreted exosomal miR-205 promotes metastasis by inducing angiogenesis.

Background: By providing oxygen, nutrients and metastatic conduits, tumour angiogenesis is essential for cancer metastasis. Cancer cell-secreted microRNAs can be packaged into exosomes and are implicated in different aspects of tumour angiogenesis. However, the underlying mechanisms are incompletely understood. Methods: The GEPIA database and in situ hybridization assay were used to analyse expression of miR-205 in ovarian tissues. Immunohistochemistry was performed to examine the relationship between miR-205 and microvessel density. Expression of circulating miR-205 was evaluated by RT-PCR and GEO database analysis. Co-culture and exosome labelling experiments were performed to assess exosomal miR-205 transfer from ovarian cancer (OC) cells to endothelial cells ECs. Exosome uptake assays were employed to define the cellular pathways associated with the endocytic uptake of exosomal miR-205. The role of exosomal miR-205 in angiogenesis was further investigated in vivo and in vitro. Western blotting and rescue experiments were applied to detect regulation of the PTEN-AKT pathway by exosomal miR-205 in ECs. Results: miR-205 was up-regulated in OC tissues, and high expression of miR-205 was associated with metastatic progression in OC patients. Moreover, miR-205 was highly enriched in cancer-adjacent ECs, and up-regulation of miR-205 correlated positively with high microvessel density in OC patients. Importantly, miR-205 was markedly enriched in the serum of OC patients, and a high level of miR-205 in circulating exosomes was associated with OC metastasis. In addition, OC-derived miR-205 was secreted into the extracellular space and efficiently transferred to adjacent ECs in an exosome-dependent manner, and the lipid raft-associated pathway plays an important role in regulating uptake of exosomal miR-205. Exosomal miR-205 from OC cells significantly promoted in vitro angiogenesis and accelerated angiogenesis and tumour growth in a mouse model. Furthermore, we found that exosomal miR-205 induces angiogenesis via the PTEN-AKT pathway. Conclusion: These findings demonstrate an exosome-dependent mechanism by which miR-205 derived from cancer cells regulates tumour angiogenesis and implicate exosomal miR-205 as a potential therapeutic target for OC.

Converting surface-oxidized cobalt phosphides into Co2(P2O7)-CoP heterostructures for efficient electrocatalytic hydrogen evolution.

Exploring noble-metal-free electrocatalysts for the hydrogen evolution reaction (HER) is a key issue in a hydrogen economy blueprint. As one of the promising candidates, transition metal phosphides unfortunately suffer from inevitable surface oxidation which obstructs active-site exposure. Herein, a facile reduction followed by a surface phosphorization is introduced to convert surface-oxidized cobalt phosphides to a Co2(P2O7)-CoP heterostructure embedded in N-doped carbon (Co2(P2O7)-CoP/NC), accomplishing an efficient HER in both acidic and alkaline electrolytes. It affords low overpotentials (η 10) of 88 and 97 mV to reach a current density of -10 mA cm-2, and small Tafel slopes of 51 and 61 mV dec-1 in 0.5 M H2SO4 and 1.0 M KOH, respectively, outperforming the parent surface-oxidized Co2P and most previously-reported Pt-free electrocatalysts. The remarkably improved electrocatalysis should be ascribed to the strong surface acidity of the Co2(P2O7) component and thereby the promoted HER kinetics on Co2(P2O7)-CoP interfaces. This work will encourage the development of cost-efficient electrocatalysts via surface engineering.