Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide CGA-N9.

Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RT‒qPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.

Bio-Based Ca2+-Selective Supramolecular Metallohydrogels with Ultra-mechanical Responsiveness, Selective Dye Adsorption, and Recycling.

Water pollution is one of the major problems that need to be solved in modern society, and there is a need to develop an effective adsorbent to purify the polluted water. In this article, three supramolecular metallohydrogels containing a three-dimensional network structure have been prepared from rosin derivatives. The supramolecular metallohydrogels have good thermal stability and maintain mechanical strength at high temperatures. Interestingly, the sodium N-(dehydroabiety1)maleamate/Ca2+ supramolecular metallohydrogels exhibit rare multi-stimulus responsiveness (mechanical vibration, temperature, pH, EDTA, etc.), especially to mechanical vibration with over 10 cycles, indicating ultra-mechanical response properties. More importantly, the unique three-dimensional network structure of the metallohydrogels can effectively adsorb cationic dyes in the wastewater. The adsorption amount and adsorption rate of this supramolecular metallohydrogels for rhodamine 6G after 48 h were at least 160.6 mg/g and 97%, respectively. The adsorption kinetic process of this metallohydrogel follows a quasi-secondary kinetic model, where the adsorption process is mainly electrostatic and weak π-π interactions. And the metallohydrogels can also be recovered by 0.5 mol/L hydrochloric acid solution desorption after adsorption of the dye. This is the first supramolecular metallohydrogel system prepared from the natural product rosin and applied to dye adsorption. This broadens the application of rosin in the field of supramolecular gel and dye adsorption and recycling.

Structure-based drug design of potential inhibitors of FBXW8, the substrate recognition component of Cullin-RING ligase 7.

FBXW8 plays an irreplaceable role in the substrate recognition of ubiquitin-dependent proteolysis, which further regulates cell cycle progression and signal transduction. However, the abnormal expression of FBXW8 triggers malignancy, inflammation, and autophagy irregulation. FBXW8 is considered as an effective therapeutic target for Cullin-RING ligase 7 (CRL7)-related cancers. Still, the lack of selective inhibitors hinders further therapeutic development and limits the exploration of its biological mechanism. This study constructed an integrated protocol that combines pharmacophore modeling, structure-based virtual screening, and Molecular Dynamic Simulation. It was then used as a screening query to identify hit compounds targeted at the substrate recognition site of FBXW8 from a large-scale compound database including 120 million compounds. Then, ten lead compounds were retrieved by using molecular docking analysis and ADMET prediction. Finally, MD simulations were performed to validate the binding stability of selected drug candidates. The result indicated that three newly obtained compounds, namely ZINC96179876, ZINC72174069, and ZINC97730272, might be potent FBXW8 inhibitors against CRL7-related cancers such as endometrial cancer.

Biodegradation efficiency and mechanism of erythromycin degradation by Paracoccus versutus W7.

Continuous and excessive usage of erythromycin results in serious environmental pollution and presents a health risk to humans. Biological treatment is considered as an efficient and economical method to remove it from the environment. In this study, a novel erythromycin-degrading bacterial strain, W7, isolated from sewage sludge was identified as Paracoccus versutus. Strain W7 degraded 58.5% of 50 mg/L erythromycin in 72 h under the optimal conditions of 35 °C, pH 7.0, and 0.1% sodium citrate with yeast powder in mineral salt medium. It completely eliminated erythromycin from erythromycin fermentation residue at concentrations of 100 and 300 mg/L within 36 and 60 h, respectively. Erythromycin esterase (EreA) was found to be involved in erythromycin metabolism in this strain and was expressed successfully. EreA could hydrolyze erythromycin, and its maximum activity occurred at pH 8.5 and 35 °C. Finally, six intermediates of erythromycin degraded by strain W7 were detected by high performance liquid chromatography mass spectrometry. Based on the novel intermediates and enzymes, we determined two possible pathways of erythromycin degradation by strain W7. This study broadened our understanding of the erythromycin catabolic processes of P. versutus and developed a feasible microbial strategy for removing erythromycin from erythromycin fermentation residue, wastewater, and other erythromycin-contaminated environments.

Field tests of crop growth using hydrothermal and spray-dried cephalosporin mycelia dregs as amendments: Utilization of nutrient and soil antibiotic resistome.

The disposal and reuse of cephalosporin mycelia dregs (CMDs) pose a great challenge to the biopharma industry, but it acts as the new source of antibiotic resistome, although agriculture intensification remains uncertain. Herein, two common cash crops (maize and soybean) were planted in the actual field, and the effects of the application of treated CMDs, chicken manure and chemical fertilizer served as control groups were both investigated according to comparison experiment. Amplicon-targeted 16S rRNA and high-throughput sequencing was analyzed for rhizosphere antibiotic resistome. Results showed that hydrothermal and spray-dried (HT + SD) CMDs could promote nutrients uptake and stabilize soil fertility indicator, and finally improved the crop yield (maximum, 119.68%). The numbers and relative abundances of total ARGs in soils were not significantly different from that of conventional fertilizer (p ＞ 0.05), but crop type marked the differences in distribution. The overall economic benefits are predicted to be around $373-745 million annually, considering its application to the whole country. HT + SD-treated CMDs can be therefore used as a high-quality and safe alternative fertilizer for agriculture use. These findings are expected to offer a fresh perspective on the application of antibiotic fermentation residue (AFR) in the future.

High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag.

The nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and inform anti-cancer drug design. Recombinant protein expression and purification of human NSDs using an E. coli expression system are notoriously challenging, but the production of pure, stable, and active NSDs is essential for further studies. To overcome production challenges, we propose a cost-efficient approach optimized to produce a high yield of NSDs using a modified E. coli expression system. We found that tagging the NSDs with a human influenza hemagglutinin (HA) tag greatly improved the quality of the recombinant NSDs, resulting in more than 95% pure, stable, and active NSD-HAs, with an increase in production yield up to 22.4-fold and up to 6.25 mg/L from LB E. coli culture, and without further purification such as ion-exchange or size-exclusion chromatography.

Identification of LEM-14 inhibitor of the oncoprotein NSD2.

The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC50 of 132 µM and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC50 of 418 µM (NSD1), IC50 of 111 µM (NSD2) and IC50 of 60 µM (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors.

Degradation of antibiotics and antibiotic resistance genes in fermentation residues by ionizing radiation: A new insight into a sustainable management of antibiotic fermentative residuals.

Antibiotic fermentative residues are categorized into hazardous wastes in China due to the existence of antibiotic resistance genes (ARGs) and residual antibiotics How to treat and manage these wastes is a new challenge. This paper investigated the treatment of erythromycin thiocyanate fermentation (EryTcF) residues using ionizing radiation technology for removing ARGs and antibiotics from the fermentation residues. The results showed that as exposed the EryTcF residues to gamma radiation, the abundance of four macrolide resistance genes (ereA, ermB, mefA and mpfB) decreased 1.0-1.3 log with 90-95% removal, and around 56% of erythromycin was removed at absorbed dose of 30 kGy and room temperature (19-22 °C). Direct action of γ-ray radiation contributed to 42-53% of ARGs removal and indirect action (radicals' reaction) was mainly responsible for erythromycin removal (84%). The positive correlation between total ARGs and Shannon index was observed. The potential ARGs-linked hosts were assigned to genera Aeromonas and Enterobacteriaceae and their abundance decreased by 36-43% at 30 kGy. Radiation has not obvious influence on the nutrient components of residues, such as protein content, suggesting that the radiation treated fermentative residues can be used as fertilizer, which is favorable for the development of recycling economy in antibiotic pharmaceutical factory. The results could provide a new insight into a sustainable management of antibiotic fermentative residuals.

Structural insights into the C-terminus of the histone-lysine N-methyltransferase NSD3 by small-angle X-ray scattering.

NSD3 is a member of six H3K36-specific histone lysine methyltransferases in metazoans. Its overexpression or mutation is implicated in developmental defects and oncogenesis. Aside from the well-characterized catalytic SET domain, NSD3 has multiple clinically relevant potential chromatin-binding motifs, such as the proline-tryptophan-tryptophan-proline (PWWP), the plant homeodomain (PHD), and the adjacent Cys-His-rich domain located at the C-terminus. The crystal structure of the individual domains is available, and this structural knowledge has allowed the designing of potential inhibitors, but the intrinsic flexibility of larger constructs has hindered the characterization of mutual domain conformations. Here, we report the first structural characterization of the NSD3 C-terminal region comprising the PWWP2, SET, and PHD4 domains, which has been achieved at a low resolution in solution by small-angle X-ray scattering (SAXS) data on two multiple-domain NSD3 constructs complemented with size-exclusion chromatography and advanced computational modeling. Structural models predicted by machine learning have been validated in direct space, by comparison with the SAXS-derived molecular envelope, and in reciprocal space, by reproducing the experimental SAXS profile. Selected models have been refined by SAXS-restrained molecular dynamics. This study shows how SAXS data can be used with advanced computational modeling techniques to achieve a detailed structural characterization and sheds light on how NSD3 domains are interconnected in the C-terminus.

The Importance of Using Exosome-Loaded miRNA for the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a major traumatic disease of the central nervous system characterized by high rates of disability and mortality. Many studies have shown that SCI can be divided into the two stages of primary and secondary injury. Primary injury leads to pathophysiological changes, while consequential injury is even more fatal, including a series of harmful reactions that expand the scope and degree of SCI. Because the pathological process of SCI is highly complex, there is still no clear and effective clinical treatment strategy. Exosomes, membrane-bound extracellular vesicles (EVs) with a diameter of 30-200 nm, have emerged as an ideal vector to deliver therapeutic molecules. At the same time, increasing numbers of studies have shown that miRNAs play a momentous role in the process of SCI. In recent studies, researchers have adopted exosomes as carriers of miRNAs with potential therapeutic effects in SCI. In this review, we summarize relevant articles describing exosomes as miRNA carriers for SCI, after which we discuss further implications and perspectives of this novel treatment modality.

Non-thermal processing of cashews: irradiation reduces allergenicity by altering the structure of Ana o 3.

Traditional thermal processing of cashews not only results in nutrient loss and harmful by-products, but also does not significantly reduce allergenicity. Irradiation could be an important non-thermal processing method to reduce cashew allergens' allergenicity and retain their nutritional properties. This study aimed to evaluate the effects of gamma irradiation processing on the structure and potential allergenicity of Ana o 3. The Ana o 3 solutions were gamma-irradiated at 0, 1, 3, 5, and 10 kGy. The structure change was monitored by Tricine-SDS-PAGE, circular dichroism spectroscopy, and fluorescence spectroscopy. The potential allergenicity was tested by immunoblotting, indirect competitive ELISA, and the human basophil KU812 degranulation assay using serum from cashew allergy patients. The results of CD spectroscopy showed that the content of α-helices decreased from 46.8% to 30.9% after 3 to 10 kGy, while the content of random coils increased from 23.7% to 33.3%. Meanwhile, a large number of hydrophobic regions were exposed, resulting in an increase in the hydrophobic surface of the protein. In terms of allergenicity, the IC50 values obtained by the competitive inhibition ELISA after irradiation increased from 0.628 to 4.054 µg mL-1, indicating that irradiation reduced the IgE binding capacity of Ana o 3, which was consistent with the results of western blotting. In addition, the basophil degranulation analysis showed that the release of IL-6, TNF-α, and histamine was decreased. It was shown that the potential allergenicity of the irradiated Ana o 3 was remarkably decreased since irradiation could mask or destroy the allergen epitopes, providing a new approach to reduce the allergenicity of cashew products.

Erythromycin fermentation residue exposure induces a short-term wave of antibiotic resistance in a soil-lettuce system.

The pattern of antibiotic resistance assembly and their unclear transfer in a soil-lettuce system render the treated erythromycin fermentation residue (EFR) land application risky. Herein, the antibiotic resistance genes (ARGs), mobile gene elements, and microbial communities were examined under erythromycin stress at three stages of lettuce growth. Erythromycin exhibited degradation rates of 99.4 % in soils for 60 d, with little uptake in the seedling tissues, reaching a 0.11-0.71 bioconcentration factor range. The EFR application rate must be limited <1 % to avoid human exposure risk. The diversity, biotic networks complexity, and edaphic ARG abundances of the rhizospheric microbial communities increased at the early stage, but returned to the control levels at the mature stage. The Planomicrobium and Pseudomonas bacterial genera were important biotic factors for erythromycin variation. Thirty-three MLSB genes (macrolide, lincosamide, and streptogramin B) conferring resistance to erythromycin were detected in soil, but only two endophytic ARGs (mphA-01 and ermX) were identified, with members of the Microvirga genus being the potential hosts. Partial least-squares path modeling suggested that erythromycin concentration was the main factor for endophytic ARGs evolution. This study highlighted the leaf endophytic ARG emergence and potential exposure human risks majorly caused by the drug traces in antibiotic fermentation residues.

Sustainable on-farm strategy for the disposal of antibiotic fermentation residue: Co-benefits for resource recovery and resistance mitigation.

Antibiotic fermentation residue is a key issue for the sustainable operation of pharmaceutical companies, and its improper disposal may cause antibiotic resistance transfer in the environment. However, little is known about the resource recycling strategy of this pharmaceutical waste. Herein, we used hydrothermal spray-dried (HT+SD) and multi-plate dryer (MD) methods to produce bio-organic fertilizers and applied them to an internal recycling model of a field trial. The concentrations of antibiotics (penicillin, cephalosporin, and erythromycin) in the bio-fertilizer, wastewater, and exhaust gas were in the range of 0.002-0.68 mg/kg, ≤ 0.35 ng/mL, and 0.03-0.89 ng/mL, respectively. The organic matter and total nitrogen, phosphorus, and potassium contents were approximately 80% and 10%, respectively. The soil bacterial community was similar among the fertilizer treatments in the same crop cultivation. A total of 233 antibiotic resistance genes (ARGs) and 43 mobile genetic elements (MGEs) were detected, including seven Rank I ARGs and five Rank II ARGs. Random forest analysis showed that gene acc(3)-Via and plasmid trb-C were biomarkers, for which the resistance and the transfer mechanisms were antibiotic inactivation and conjugation, respectively. The results imply that AFR recycling disposal mode is a promising prospect for pharmaceutical waste management.

Environmental factors induced macrolide resistance genes in composts consisting of erythromycin fermentation residue, cattle manure, and maize straw.

With the growing concerns about antibiotic resistance, it is more and more important to prevent the environmental pollution caused by antibiotic fermentation residues. In this study, composted erythromycin fermentation residue (EFR) with the mixture of cattle manure and maize straw at ratios of 0:10 (CK), 1:10 (T1), and 3:10 (T2) explores the effects on physicochemical characteristics, mobile genetic elements (MGEs), and antibiotic resistance genes (ARGs). Results reflected that the addition of EFR reduced the carbon/nitrogen ratio of each compost and improved the piles' temperature, which promoted the composting process. However, the contents of Na+, SO42-, and erythromycin were also significantly increased. After 30 days of composting, the degradation rates of erythromycin in CK, T1, and T2 were 72.7%, 20.3%, and 37.1%, respectively. Meanwhile, the total positive rates for 26 detected ARGs in T1 and T2 were 65.4%, whereas that of CK was only 23.1%. Further analysis revealed that ARGs responsible for ribosomal protection, such as ermF, ermT, and erm(35), dominated the composts of T1 and T2, and most were correlated with IS613, electrical conductivity (EC), nitrogen, and Zn2+. Above all, adding EFR helps to improve the nutritional value of composts, but the risks in soil salinization and ARG enrichment caused by high EC and erythromycin content should be further investigated and eliminated.

CD44 connects autophagy decline and ageing in the vascular endothelium.

The decline of endothelial autophagy is closely related to vascular senescence and disease, although the molecular mechanisms connecting these outcomes in vascular endothelial cells (VECs) remain unclear. Here, we identify a crucial role for CD44, a multifunctional adhesion molecule, in controlling autophagy and ageing in VECs. The CD44 intercellular domain (CD44ICD) negatively regulates autophagy by reducing PIK3R4 and PIK3C3 levels and disrupting STAT3-dependent PtdIns3K complexes. CD44 and its homologue clec-31 are increased in ageing vascular endothelium and Caenorhabditis elegans, respectively, suggesting that an age-dependent increase in CD44 induces autophagy decline and ageing phenotypes. Accordingly, CD44 knockdown ameliorates age-associated phenotypes in VECs. The endothelium-specific CD44ICD knock-in mouse is shorter-lived, with VECs exhibiting obvious premature ageing characteristics associated with decreased basal autophagy. Autophagy activation suppresses the premature ageing of human and mouse VECs overexpressing CD44ICD, function conserved in the CD44 homologue clec-31 in C. elegans. Our work describes a mechanism coordinated by CD44 function bridging autophagy decline and ageing.

Rice washing drainage (RWD) embedded in poly(vinyl alcohol)/sodium alginate as denitrification inoculum for high nitrate removal rate with low biodiversity.

Immobilization technology with low maintenance is a promising alternative to enhance nitrate removal from water. In this study, washing rice drainage (RWD) was immobilized by poly(vinyl alcohol)/sodium alginate (PVA/SA) to obtain RWD-PVA/SA gel beads as inoculum for denitrification. When initial nitrate concentration was 50 mg N/L, nitrate was effectively removed at rates of 50-600 mg/(L∙d) using acetate as carbon source (C/N = 1.25). Arrhenius activation energy (Ea) of nitrate oxidoreductase was 28.64 kJ/mol for the RWD-PVA/SA gel beads. Temporal and spatial variation in microbial community structures were revealed along with RWD storage and in the reactors by Illumina high-throughput sequencing technology. RWD-PVA/SA gel beads has a simple (operational taxonomic units (OTUs) ã€ˆ100). Dechloromonas, Pseudomonas, Flavobacterium and Acidovorax were the most four dominant genera in the denitrification reactors inoculated with RWD-PVA/SA gel beads. This study provides an inoculum for denitrification with high nitrate removal performance and simple microbial community structures.

Wheat Amylase Trypsin Inhibitors Aggravate Intestinal Inflammation Associated with Celiac Disease Mediated by Gliadin in BALB/c Mice.

Celiac disease (CD) is an autoimmune intestinal disorder caused by the ingestion of gluten in people who carry the susceptible gene. In current celiac disease research, wheat gluten is often the main target of attention, neglecting the role played by non-gluten proteins. This study aimed to describe the effects of wheat amylase trypsin inhibitors (ATI, non-gluten proteins) and gliadin in BALB/c mice while exploring the further role of relevant adjuvants (cholera toxin, polyinosinic: polycytidylic acid and dextran sulfate sodium) intervention. An ex vivo splenocyte and intestinal tissue were collected for analysis of the inflammatory profile. The consumption of gliadin and ATI caused intestinal inflammation in mice. Moreover, the histopathology staining of four intestinal sections (duodenum, jejunum, terminal ileum, and middle colon) indicated that adjuvants, especially polyinosinic: polycytidylic acid, enhanced the villi damage and crypt hyperplasia in co-stimulation with ATI and gliadin murine model. Immunohistochemical results showed that tissue transglutaminase and IL-15 expression were significantly increased in the jejunal tissue of mice treated with ATI and gliadin. Similarly, the expression of inflammatory factors (TNF-α, IL-1ß, IL-4, IL-13) and Th1/Th2 balance also showed that the inflammation response was significantly increased after co-stimulation with ATI and gliadin. This study provided new evidence for the role of wheat amylase trypsin inhibitors in the pathogenesis of celiac disease.

CUR5g, a novel autophagy inhibitor, exhibits potent synergistic anticancer effects with cisplatin against non-small-cell lung cancer.

Autophagy, a highly conserved degradation process of eukaryotic cells, has been proven to be closely related to chemoresistance and metastasis of non-small-cell lung cancer (NSCLC). Autophagy inhibitors, such as chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), has been shown to mediate anticancer effects in preclinical models, especially when combined with chemotherapy. However, the vast majority of autophagy inhibitors, including CQ and HCQ, actually disrupt lysosomal or/and possibly non-lysosomal processes other than autophagy. It is therefore of great significance to discover more specific autophagy inhibitors. In this study, after screening a series of curcumin derivatives synthesized in our laboratory, we found that (3E,5E)-1-methyl-3-(4-hydroxybenzylidene)-5-(3-indolymethylene)-piperidine-4-one (CUR5g) selectively inhibited autophagosome degradation in cancer cells by blocking autophagosome-lysosome fusion. CUR5g did not affect the lysosomal pH and proteolytic function, nor did it disturb cytoskeleton. CUR5g blocked the recruitment of STX17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, to autophagosomes via a UVRAG-dependent mechanism, resulting in the inability of autophagosomes to fuse with lysosomes. CUR5g alone did not induce apoptosis and necrosis of A549 cells, but significantly inhibited the mobility and colony formation of A549 cells. More excitingly, CUR5g showed no obvious toxicity to normal HUVECs in vitro or mice in vivo. CUR5g enhances the cisplatin sensitivity of A549 cells and effectively inhibited autophagy in tumor tissues in vivo. Collectively, our study identified a new late-stage autophagy inhibitor and provided a novel option for NSCLC treatment, particular when combined with cisplatin.

Assessing the effect of treated erythromycin fermentation residue on antibiotic resistome in soybean planting soil: In situ field study.

As a by-product in the pharmaceutical industry, antibiotic fermentation residue is expected to be able to be utilized after effectively removing the antibiotics. However, evaluation of the effect of fermentation residue application on soil, especially the in situ environmental consequences considering not only the antibiotic resistance gene (ARG) abundance but also the resistome risk, has still not been sufficiently evaluated. Herein, the impact of treated erythromycin fermentation residue (EFR) on the resistome and risk score in soybean planting soil was investigated. Treated EFR application with dosages of 3750 kg (EFR250) and 7500 kg (EFR500) per hm2 soil did not increase the diversity (Shannon index, 2.84-3.38) or relative abundance (0.086-0.142 copies/16S rRNA gene) of the soil resistome compared with the Control (CK: 2.92-3.2, 0.088-0.096 copies/16S rRNA gene). Soil resistome risk scores calculated by metagenomic assembly, showing the dissemination potential of ARGs, ranged from 22.9 to 25.0, and were also not significantly different between treated EFR amended soil and the Control. Notably, the diversity of the resistome increased at the sprout stage (Mann-Whitney U test, P < 0.05) and the abundance of some ARG types (macrolide-lincosamide-streptogramin, aminoglycoside and tetracycline, etc.) shifted along the course of soybean growth (Kruskal-Wallis test, P < 0.05). Structural equation model analysis showed that the soybean growth period affected the composition of ARGs by affecting the microbial community, which was further supported by Procrustes analysis (P < 0.05) and metagenomic binning. Our findings emphasized that soil ARG abundance and resistome risk did not increase during one-time field application of treated EFR at the studied dosage. Comprehensive consideration including resistome risk and multiple influencing factors also should be given for further assessment of fermentation residue application.

Polyethylenimine-Modified Mesoporous Silica Nanoparticles Induce a Survival Mechanism in Vascular Endothelial Cells via Microvesicle-Mediated Autophagosome Release.

Surface-modified mesoporous silica nanoparticles (MSNs) have attracted more and more attention as promising materials for biomolecule delivery. However, the lack of detailed evaluation relevant to the potential cytotoxicity of these MSNs is still a major obstacle for their applications. Unlike the bare MSNs and amino- or liposome-modified MSNs, we found that polyethylenimine-modified MSNs (MSNs-PEI) had no obvious toxicity to human umbilical vein endothelial cells (HUVECs) at the concentrations up to 100 µg/mL. However, MSNs-PEI induced autophagosomes accumulation by blocking their fusion with lysosomes, an essential mechanism for the cytotoxicity of many nanoparticles (NPs). Thus, we predicted that an alternative pathway for autophagosome clearance exists in HUVECs to relieve autophagic stress induced by MSNs-PEI. We found that MSNs-PEI prevented STX17 loading onto autophagosomes instead of influencing lysosomal pH or proteolytic activity. MSNs-PEI induced the structural alternation of the cytoskeleton but did not cause endoplasmic reticulum stress. The accumulated autophagosomes were released to the extracellular space via microvesicles (MVs) when the autophagic degradation was blocked by MSNs-PEI. More importantly, blockade of either autophagosome formation or release caused the accumulation of damaged mitochondria and excessive ROS production in the MSNs-PEI-treated HUVECs, which in turn led to cell death. Thus, we propose here that the MV-mediated autophagosome release, a compensation mechanism, allows the vascular endothelial cell survival when the degradation of autophagosomes is blocked by MSNs-PEI. Accordingly, promoting the release of accumulated autophagosomes may be a protective strategy against the endothelial toxicity of NPs.