TLR Engagement Induces an Alternate Pathway for BCR Signaling that Results in PKCδ Phosphorylation.

Recently, we reported that preexposure of B cells to IL-4 induced an alternate, signalosome-independent BCR signaling pathway leading to protein kinase C (PKC)δ phosphorylation (pTyr311), which occurs in the membrane compartment. This is considered to represent a form of receptor crosstalk and signal integration. Unlike the classical BCR signaling pathway, Lyn kinase is indispensable for BCR-induced downstream events in the alternate pathway. Our previous report that alternate BCR signaling leading to ERK phosphorylation is triggered by LPS and PAM3CSK4 (much like IL-4) raises the possibility that other signaling outcomes such as PKCδ phosphorylation might be similarly affected. To explore the range of mediators capable of producing an alternate pathway for BCR signaling, we examined PKCδ translocation and phosphorylation in LPS- and PAM3CSK4-treated B cells stimulated by anti-Ig. We found that LPS and PAM3CSK4 alter the signaling pathway used by the BCR to produce PKCδ phosphorylation. As with IL-4, elements of the signalosome are not needed for PKCδ phosphorylation when BCR triggering occurs after LPS and PAM3CSK4. However, with LPS and PAM3CSK4, anti-Ig-induced phosphorylation of PKCδ takes place in the cytosol, in contrast to the IL-4-induced alternate pathway, wherein PKCδ phosphorylation occurs in the membrane. Furthermore, the BCR signaling pathway induced by LPS and PAM3CSK4 differs from that induced by IL-4 by not requiring Lyn. Thus, an alternate, signalosome-independent BCR signaling pathway for PKCδ phosphorylation is induced by TLR agonists but differs in important ways from the alternate pathway induced by IL-4.

Pangenome analysis of Shewanella xiamenensis revealed important genetic traits concerning genetic diversity, pathogenicity and antibiotic resistance.

BACKGROUND: Shewanella xiamenensis, widely distributed in natural environments, has long been considered as opportunistic pathogen. Recently, significant changes in the resistance spectrum have been observed in S. xiamenensis, due to acquired antibiotic resistance genes. Therefore, a pan-genome analysis was conducted to illuminate the genomic changes in S. xiamenensis. RESULTS: Phylogenetic analysis revealed three major clusters and three singletons, among which close relationship between several strains was discovered, regardless of their host and niches. The "open" genomes with diversity of accessory and strain-specific genomes took advantage towards diversity environments. The purifying selection pressure was the main force on genome evolution, especially in conservative genes. Only 53 gene families were under positive selection pressure. Phenotypic resistance analysis revealed 21 strains were classified as multi-drug resistance (MDR). Ten types of antibiotic resistance genes and two heavy metal resistance operons were discovered in S. xiamenensis. Mobile genetic elements and horizontal gene transfer increased genome diversity and were closely related to MDR strains. S. xiamenensis carried a variety of virulence genes and macromolecular secretion systems, indicating their important roles in pathogenicity and adaptability. Type IV secretion system was discovered in 15 genomes with various sequence structures, indicating it was originated from different donors through horizontal gene transfer. CONCLUSIONS: This study provided with a detailed insight into the changes in the pan-genome of S. xiamenensis, highlighting its capability to acquire new mobile genetic elements and resistance genes for its adaptation to environment and pathogenicity to human and animals.

Splenic Dendritic Cells Survey Red Blood Cells for Missing Self-CD47 to Trigger Adaptive Immune Responses.

Sheep red blood cells (SRBCs) have long been used as a model antigen for eliciting systemic immune responses, yet the basis for their adjuvant activity has been unknown. Here, we show that SRBCs failed to engage the inhibitory mouse SIRPα receptor on splenic CD4(+) dendritic cells (DCs), and this failure led to DC activation. Removal of the SIRPα ligand, CD47, from self-RBCs was sufficient to convert them into an adjuvant for adaptive immune responses. DC capture of Cd47(-/-) RBCs and DC activation occurred within minutes in a Src-family-kinase- and CD18-integrin-dependent manner. These findings provide an explanation for the adjuvant mechanism of SRBCs and reveal that splenic DCs survey blood cells for missing self-CD47, a process that might contribute to detecting and mounting immune responses against pathogen-infected RBCs.

A meta-analysis of randomized controlled trials evaluating the effectiveness of fecal microbiota transplantation for patients with irritable bowel syndrome.

OBJECTIVE: Multiple randomized controlled trials (RCTs) have investigated the efficacy of fecal microbiota transplantation (FMT) for irritable bowel syndrome (IBS), but have yielded inconsistent results. We updated the short-term and long-term efficacy of FMT in treating IBS, and performed a first-of-its-kind exploration of the relationship between gut microbiota and emotions. METHODS: We conducted a comprehensive search of PubMed, Embase, Web of Science, and the Cochrane Library using various search strategies to identify all eligible studies. The inclusion criteria for data extraction were randomized controlled trials (RCTs) that investigated the efficacy of fecal microbiota transplantation (FMT) compared to placebo in adult patients (≥ 18 years old) with irritable bowel syndrome (IBS). A meta-analysis was then performed to assess the summary relative risk (RR) and corresponding 95% confidence intervals (CIs). RESULTS: Out of 3,065 potentially relevant records, a total of 10 randomized controlled trials (RCTs) involving 573 subjects met the eligibility criteria for inclusion in the meta-analysis. The meta-analyses revealed no significant differences in short-term (12 weeks) (RR 0.20, 95% CI -0.04 to 0.44), long-term (52 weeks) global improvement (RR 1.38, 95% CI 0.87 to 2.21), besides short-term (12 weeks) (SMD - 48.16, 95% CI -102.13 to 5.81, I2 = 90%) and long-term (24 weeks) (SMD 2.16, 95% CI -60.52 to 64.83, I2 = 68%) IBS-SSS. There was statistically significant difference in short-term improvement of IBS-QoL (SMD 10.11, 95% CI 0.71 to 19.51, I2 = 82%), although there was a high risk of bias. In terms of long-term improvement (24 weeks and 54 weeks), there were no significant differences between the FMT and placebo groups (SMD 7.56, 95% CI 1.60 to 13.52, I2 = 0%; SMD 6.62, 95% CI -0.85 to 14.08, I2 = 0%). Sensitivity analysis indicated that there were visible significant effects observed when the criteria were based on Rome IV criteria (RR 16.48, 95% CI 7.22 to 37.62) and Gastroscopy (RR 3.25, 95%CI 2.37 to 4.47), Colonoscopy (RR 1.42, 95% CI 0.98 to 2.05). when using mixed stool FMT based on data from two RCTs, no significant difference was observed (RR 0.94, 95% CI 0.66 to -1.34). The remission of depression exhibited no significant difference between the FMT and placebo groups at the 12-week mark (SMD - 0.26, 95% CI -3.09 to 2.58), and at 24 weeks (SMD - 2.26, 95% CI -12.96 to 8.45). Furthermore, major adverse events associated with FMT were transient and self-limiting. DISCUSSION: Based on the available randomized controlled trials (RCTs), the current evidence does not support the efficacy of FMT in improving global IBS symptoms in the long term. The differential results observed in subgroup analyses raise questions about the accurate identification of suitable populations for FMT. Further investigation is needed to better understand the reasons behind these inconsistent findings and to determine the true potential of FMT as a treatment for IBS.

Molecular characterization of extensively drug-resistant hypervirulent Pseudomonas aeruginosa isolates in China.

BACKGROUND: Recently, extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) isolates have been increasingly detected and posed great challenges to clinical anti-infection treatments. However, little is known about extensively resistant hypervirulent P. aeruginosa (XDR-hvPA). In this study, we investigate its epidemiological characteristics and provide important basis for preventing its dissemination. METHODS: Clinical XDR-PA isolates were collected from January 2018 to January 2023 and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; antibiotic susceptibility testing was performed by broth microdilution method, and minimum inhibitory concentrations (MICs) were evaluated. Virulence was evaluated using the Galleria mellonella infection model; molecular characteristics, including resistance genes, virulence genes, and homology, were determined using whole-genome sequencing. RESULTS: A total of 77 XDR-PA strains were collected; 47/77 strains were XDR-hvPA. Patients aged > 60 years showed a significantly higher detection rate of XDR-hvPA than of XDR-non-hvPA. Among the 47 XDR-hvPA strains, 24 strains carried a carbapenemase gene, including blaGES-1 (10/47), blaVIM-2 (6/47), blaGES-14 (4/47), blaIMP-45 (2/47), blaKPC-2 (1/47), and blaNDM-14 (1/47). ExoU, exoT, exoY, and exoS, important virulence factors of PA, were found in 31/47, 47/47, 46/47, and 29/47 strains, respectively. Notably, two XDR-hvPA simultaneously co-carried exoU and exoS. Six serotypes (O1, O4-O7, and O11) were detected; O11 (19/47), O7 (13/47), and O4 (9/47) were the most prevalent. In 2018-2020, O4 and O7 were the most prevalent serotypes; 2021 onward, O11 (16/26) was the most prevalent serotype. Fourteen types of ST were detected, mainly ST235 (14/47), ST1158 (13/47), and ST1800 (7/47). Five global epidemic ST235 XDR-hvPA carried blaGES and showed the MIC value of ceftazidime/avibactam reached the susceptibility breakpoint (8/4 mg/L). CONCLUSIONS: The clinical detection rate of XDR-hvPA is unexpectedly high, particularly in patients aged > 60 years, who are seemingly more susceptible to contracting this infection. Clonal transmission of XDR-hvPA carrying blaGES, which belongs to the global epidemic ST235, was noted. Therefore, the monitoring of XDR-hvPA should be strengthened, particularly for elderly hospitalized patients, to prevent its spread.

Study on the Synergistic Mechanism of Photodynamic Therapy Combined With Ferroptosis Inducer to Induce Ferroptosis in Cholangiocarcinoma.

BACKGROUND: Photodynamic therapy (PDT) induced lipid peroxidation reaction can lead to necrosis and apoptosis of extrahepatic cholangiocarcinoma (ECC) cells, reducing the tumor load. However, the depth of action of PDT is shallow, and its therapy efficacy is weak, making it difficult to achieve eradication even with multiple treatments. OBJECTIVES: This study aims to investigate the mechanism and main pathways of ferroptosis in cholangiocarcinoma under Hematoporphyrin-mediated photodynamic therapy, and to compare the effects of different ferroptosis inducers on photodynamic therapy-induced ferroptosis in cholangiocarcinoma. To provide an experimental basis for selecting appropriate ferroptosis-inducing agents and synergizing with photodynamic therapy during the clinical perioperative period. METHODS: The Cell Counting Kit-8 (CCK-8) was used to examine the cytotoxicity of cholangiocarcinoma cells following PDT. Flow cytometry was used to detect apoptotic cell percentage and cell cycle changes to assess the enhanced photodynamic production of reactive oxygen species (ROS) by different ferroptosis inducers, confocal imaging was used to de-assay ROS content. Western blot analysis was employed to detect the expression of GPX4 、FSP1、ASCL4 and SLC7A11. Furthermore, a fluorescence spectrophotometric assay was used to quantify the alterations in lipid peroxides (MDA, LPO, GSH, and Fe2+). RESULTS: The combination of PDT with Lenvatinib or Erastin resulted in increased ROS levels, and decreased GSH content, tumor cells were inhibited in the G2 phase, and the proportion of apoptotic cells increased. Additionally, GPX4, FSP1, and SLC7A11 protein expression decreased, whereas ASCL4 increased This was accompanied by heightened levels of Fe2+, LPO, and MDA. Induction of the ferroptosis pathway was observed to enhance the therapeutic efficacy of PDT. CONCLUSION: Our findings suggest that Erastin or Lenvatinib can enhance the induction of ferroptosis in cholangiocarcinoma cells by photodynamic therapy by increasing intracellular ROS and inhibiting intracellular antioxidant pathways.

Paeoniflorin alleviates TGF-ß2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

BACKGROUND: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-ß2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. METHODS: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-ß2 (5 ng/mL). PAE (300 µM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-ß2 for 22 h. SB-431542, a TGF-ß receptor inhibitor (10 µM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-ß2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. RESULTS: PAE attenuated TGF-ß2-induced oxidative stress and suppressed TGF-ß2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-ß2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-ß2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. CONCLUSION: PAE alleviates TGF-ß2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Clonal transmission of polymyxin B-resistant hypervirulent Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaKPC-2 in a tertiary hospital in China.

BACKGROUND: The prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak. RESULTS: The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored blaKPC-2, blaNDM-1, and the virulence-related genes prmpA, prmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8-20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying blaKPC-2, blaNDM-1, blaSHV-12, blaLAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance. CONCLUSIONS: Polymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Signal Integration by Translocation and Phosphorylation of PKCδ in the B Cell Alternate Pathway.

B cell signaling for activation via the BCR occurs as an isolated event only in vitro; in real life, BCR signaling takes place within a complex milieu that involves interactions with agents that trigger additional receptors. Chief among these is IL-4. We have shown that BCR signaling is reprogrammed by IL-4 receptor engagement and that this reprogramming involves creation of a new, signalosome-independent, Lyn-dependent alternate signaling pathway in B cells isolated from BALB/cByJ mice. A unique aspect of the alternate pathway is protein kinase Cδ (PKCδ) phosphorylation. In dissecting this pathway, we unexpectedly found that Lyn is associated with IL-4Rα, that IL-4 induces Lyn activation, and that Lyn immunoprecipitated from IL-4-treated B cells capably phosphorylates PKCδ in a cell-free system. However, PKCδ phosphorylation does not occur in the absence of BCR triggering in vivo. This raised the question of why IL-4 alone failed to produce PKCδ phosphorylation. We considered the possibility that Lyn and PKCδ may be spatially separated. As expected, before any treatment, Lyn is located primarily in the membrane fraction, whereas PKCδ is located mainly in the cytosol fraction. However, when anti-Ig follows IL-4 treatment, PKCδ is found in the membrane fraction and phosphorylated. This translocation of PKCδ to the membrane fraction is not affected by loss of Lyn, although PKCδ phosphorylation requires Lyn. Thus, PKCδ phosphorylation through the alternate pathway represents the result of signal integration, whereby neither IL-4 nor anti-Ig working alone produces this outcome, but together they achieve this result by Lyn activation (IL-4) and PKCδ translocation (IL-4 followed by anti-Ig).

Metagenomic analysis reveals the short-term influences on conjugation of blaNDM-1 and microbiome in hospital wastewater by silver nanoparticles at environmental-related concentration.

Hospital wastewater contains large amounts of antibiotic-resistant bacteria and serves as an important reservoir for horizontal gene transfer (HGT). However, the response of the microbiome in hospital wastewater to silver remains unclear. In this study, the short-term impacts of silver on the microbiome in hospital wastewater were investigated by metagenome next-generation sequencing. The influence of silver on the conjugation of plasmid carrying blaNDM-1 was further examined. Our results showed that in hospital wastewater, high abundances of antibiotic resistance genes (ARGs) were detected. The distribution tendencies of certain ARG types on chromosomes or plasmids were different. Clinically important ARGs were identified in phage-like contigs, indicating potential transmission via transduction. Pseudomonadales, Enterobacterales, and Bacteroidales were the major ARG hosts. Mobile genetic elements were mainly detected in plasmids and associated with various types of ARGs. The binning approach identified 29 bins that were assigned to three phyla. Various ARGs and virulence factors were identified in 14 and 11 bins, respectively. MetaCHIP identified 49 HGT events. The transferred genes were annotated as ARGs, mobile genetic elements, and functional genes, and they mainly originated from donors belonging to Bacteroides and Pseudomonadales. In addition, 20 nm AgNPs reduced microbial diversity and enhanced the relative abundance of Acinetobacter. The changes induced by 20 nm AgNPs included increases in the abundances of ARGs and genes involved lipid metabolism pathway. Conjugation experiments showed that Ag+ and 20 nm AgNPs caused 2.38-, 3.31-, 4.72-, and 4.57-fold and 1.46-, 1.61-, 3.86-, and 2.16-fold increases in conjugation frequencies of plasmid with blaNDM-1 at 0.1, 1, 10, and 100 µg/L, respectively. Our findings provide insight into the response of the microbiome in hospital wastewater to silver, emphasize the adaptation capability of Acinetobacter inhabiting hospitals against adverse environments, and highlight the promotion of silver for antibiotic resistance.

Emergence of polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae from an individual in the community with asymptomatic bacteriuria.

BACKGROUND: The heteroresistance of polymyxin B, a last-resort antibiotic used to treat many serious bacterial infections, may lead to antibiotic treatment failure. However, polymyxin B-heteroresistant isolates are rare in individuals living in the community. We report a polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae (hvKP) isolate from an individual in the community with asymptomatic bacteriuria. RESULTS: The NYTJ35 isolate had multiple virulence genes that encoded a mucoid phenotype regulator (rmpA), aerobactin (iucABCD-iutA), salmochelin (iroBCDN), yersiniabactin (irp1-2 and ybtAEPQSTUX), and a truncated rmpA2. Infection of galleria mellonella larvae indicated the isolate was hypervirulent. Antimicrobial susceptibility testing showed it was susceptible to all tested antibiotics except polymyxin B. The proportion of surviving bacteria was 1.2 × 10- 7 based on the population analysis profile (PAP) method, suggesting the presence of polymyxin B heteroresistance. The isolate was not hypermucoviscous, but it was a strong biofilm producer. It had capsular serotype K1 and belonged to sequence type 23 (ST23). The isolate also had the D150G substitution in phoQ, which is known to confer polymyxin B resistance. CONCLUSIONS: We identified the co-occurrence of hypervirulence and polymyxin B heteroresistance in a K. pneumoniae isolate from an individual with asymptomatic bacteriuria. We suggest the use of increased screening for hvKP in individuals living in the community.

Distribution, characterization, and antibiotic resistance of hypervirulent Klebsiella pneumoniae isolates in a Chinese population with asymptomatic bacteriuria.

BACKGROUND: Asymptomatic bacteriuria (ASB) frequently occurs among all ages and may develop into urinary tract infections (UTIs). Hypervirulent Klebsiella pneumoniae (hvKP) has become a new threat to human health. In our study, we aimed to investigate the epidemiological characteristics of hvKP in population with ASB. RESULTS: A total of 61 K. pneumoniae isolates were collected from 7530 urine samples between October and December 2020. The strains were sensitive to most of the antimicrobial agents tested, but a polymyxin resistant strain was found (MIC>16 µg/mL). Three serotypes were detected, including K1 (16.4%, 10/61), K5 (1.6%, 1/61) and K57 (3.2%, 2/61). Four strains (KPNY9, KPNY31, KPNY40, and KPNY42) carried a combination of two or more hypervirulent markers (peg-344, iroB, iucA, prmpA, and prmpA2), and their survival rates after Galleria mellonella infection were lower than those of the other strains (40.0 vs. 70.0%), suggesting that they were hvKP. These hvKP strains with lower biofilm forming ability than classical K. pneumoniae (0.2625 ± 0.0579 vs. 0.6686 ± 0.0661, P = 0.033) were identified as belonging to K2-ST65, K2-ST86, K57-ST592, and K2-ST5559 (a new ST type). KPNY31 (ST5559) shared a close genetic relationship with KPNY42 (ST86) and other ST86 isolates, which have been detected in both nosocomial and community-acquired infections. CONCLUSIONS: The hvKP with relatively weak biofilm formation was detected in a population with ASB, which was more likely to cause bacteremia and serious consequences. A novel sequence type (ST5559) hvKP derived from ST86 was found. Therefore, hvKP should be monitored in the population with ASB.

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.

BACKGROUND: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. RESULTS: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

Protease or Clostridium butyricum addition to a low-protein diet improves broiler growth performance.

Low-protein (LP) feeds are used in the poultry industry to combat the increasing consumption of protein resources and reduce environmental pollution caused by excessive nitrogen excretion. Dietary supplementation of protease or Clostridium butyricum increases the growth performance of broilers; however, it is unclear whether they counteract the negative effects of LP diets. The effects of protease and C. butyricum on growth performance, intestinal morphology, anti-oxidant capacity, anti-inflammatory response, and microbial community of broilers have not been studied extensively. Here, 450 healthy 1-day-old Cobb500 broilers were allocated to five groups, according to different diets: basal diet (Control); LP diet (LP; 2% less crude protein than the control); LP diet + 200 g/t HuPro protease (LPH); LP diet + 1.0 × 109 CFU/t C. butyricum (LPC); and basal diet + 200 g/t oxytetracycline (Antibiotic). Supplementing both C. butyricum and protease improved the growth performance of broilers. The supplementation of HuPro protease under low-protein conditions could achieve a breeding effect similar to that of the positive control (Antibiotic). Supplementing C. butyricum could maintain intestinal barrier function, alleviate the inflammatory response, and increase ileal and cecal short-chain fatty acid concentrations. Both C. butyricum and protease altered the bacterial diversity in the cecum, increased Bacteroidetes abundance, and resulted in higher abundance of Rikenellaceae RC9 gut spp. and lower abundance of Alistipes spp. in broilers. This study demonstrates the positive effects of proteases and C. butyricum on broilers and serves as a reference for the selection of appropriate supplementation for broilers in the poultry industry. KEY POINTS: • Low-protein diet had a negative effect on growth performance of broilers. • Protease significantly reduced feed conversion rate. • Clostridium butyricum had positive effects on broilers.

Characteristics of Staphylococcus aureus small colony variants isolated from wound specimen of a tertiary care hospital in China.

BACKGROUND: Small colony variants (SCVs) of Staphylococcus aureus (S. aureus) frequently lead to chronic and recurrent infections, but they are always ignored and there are few researches on their clinical isolates. We intended to investigate the prevalence and characteristics of S. aureus SCVs. METHODS: None-duplicated S. aureus strains isolated from wound samples were collected from January 2018 to December 2020. The characteristics (i.e. colony morphology, growth rate, coagulase, biofilm formation, and pathogenic characteristics), antimicrobial susceptibilities, and resistance mechanisms of SCVs were also investigated. The genetic background of SCVs was analyzed through staphylococcal protein A (SPA) typing, sequence typing, and pulse field gel electrophoresis (PFGE). RESULTS: Three SCVs were screened from 278 S. aureus strains (1.1%). They formed pinpoint white colonies on blood agar plates with weak hemolysis. The reproduction speed in liquid medium was very slow for SCVs strains. The coagulase weakened or disappeared, and the ability to form biofilm varied greatly. Only slight inflammation was triggered when wound infected. The SPA typing was t2592, t233, and t023, and the sequence typing was ST88, ST239, and ST965, respectively. The PFGE revealed three SCVs were singletons. CONCLUSIONS: The rate of SCVs in wound sample is low in our hospital, and the formation is associated with the usage of antimicrobial. SCVs grow slowly, and their colony morphology and biochemical characteristics are significantly different from classic S. aureus. SCVs may cause chronic infection and weak inflammation. SCVs form in resistant or susceptible strains, and there is no clonal epidemic in this hospital.

Determination of xanthine using a ratiometric fluorescence probe based on boron-doped carbon quantum dots and gold nanoclusters.

A dual-emission ratiometric fluorescent sensing system based on boron-doped carbon quantum dots (B-CQDs) and gold nanoclusters (AuNCs) has been developed for the determination of xanthine. The blue fluorescence of B-CQDs at 445 nm is then reduced by the AuNCs through the inner filter effect (IFE) under a single excitation wavelength of 370 nm. By the catalysis of xanthine oxidase (XOD), xanthine is oxidized by oxygen dissolved in the solution to produce H2O2. The horseradish peroxidase (HRP) catalyzes H2O2 to generate hydroxyl radicals, which can quench the fluorescence of AuNCs, leading to the recovery of the fluorescence of B-CQDs. Based on the relationship between the fluorescence intensity ratio (F445/F665) and the concentration of xanthine, the designed method exhibits a good linearity range of 1.2-500.0 µmol L -1 and a limit of detection of 0.37 µmol L -1. The ratiometric fluorescent is applied to determine xanthine in human urine samples. Good recoveries of spiked samples in the range 99.2-105.0% are obtained by the proposed assay, with relative standard deviations (RSD) ranging from 0.9 to 2.6%.

Spleen tyrosine kinase facilitates neutrophil activation and worsens long-term neurologic deficits after spinal cord injury.

BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.

Characterization of fosfomycin resistance and molecular epidemiology among carbapenem-resistant Klebsiella pneumoniae strains from two tertiary hospitals in China.

BACKGROUND: Fosfomycin has been proven to be a vital choice to treat infection caused by multidrug resistance bacteria, especially carbapenem-resistant Klebsiella pneumoniae (CRKP). However, fosfomycin resistant cases has been reported gradually. In this study, we reported the fosfomycin-resistant rate in CRKP strains and further revealed the molecular mechanisms in resistance gene dissemination. RESULTS: A total of 294 non-duplicated CRKP strains were collected. And 55 fosfomyin-resistant strains were detected, 94.5% of which were clustered to sequence type (ST) 11 by PCR followed up sequencing. PFGE further revealed two major groups and four singletons. The positive rates of genes responsible to fosfomycin and carbapenem resistance were 81.8% (fosA3), 12.7% (fosA5) and 94.5% (blaKPC-2), respectively. Genomic analysis confirmed insertion sequence (IS) 26 was the predominant structure surrounding fosA3. The fosA3 genes in six isolates were located on plasmids which were able to transfer to E. coli J53 recipient cells by means of conjugation. CONCLUSIONS: Although the resistant rate of CRKP to fosfomycin is relatively low in our area, considering its gene is located on transferrable plasmid and inserted in IS structure, continuous monitoring is still needed.

Antifungal Activity and Action Mechanism Study of Coumarins from Cnidium monnieri Fruit and Structurally Related Compounds.

The increasing resistance of plant diseases caused by phytopathogenic fungi highlights the need for highly effective and environmentally benign agents. The antifungal activities of Cnidium monnieri fruit extracts and five isolated compounds as well as structurally related coumarins against five plant pathogenic fungi were evaluated. The acetone extract, which contained the highest amount of five coumarins, showed strongest antifungal activity. Among the coumarin compounds, we found that 4-methoxycoumarin exhibited stronger and broader antifungal activity against five phytopathogenic fungi, and was more potent than osthol. Especially, it could significantly inhibit the growth of Rhizoctonia solani mycelium with an EC50 value of 21 µg mL-1 . Further studies showed that 4-methoxycoumarin affected the structure and function of peroxisomes, inhibited the ß-oxidation of fatty acids, decreased the production of ATP and acetyl coenzyme A, and then accumulated ROS by damaging MMP and the mitochondrial function to cause the cell death of R. solani mycelia. 4-Methoxycoumarin presented antifungal efficacy in a concentration- dependent manner in vivo and could be used to prevent the potato black scurf. This study laid the foundation for the future development of 4-methoxycournamin as an alternative and friendly biofungicide.

The Antifungal Mechanism of Isoxanthohumol from Humulus lupulus Linn.

Humulus lupulus Linn. is a traditional medicinal and edible plant with several biological properties. The aims of this work were: (1) to evaluate the in vitro antifungal activity of H. lupulus ethanolic extract; (2) to study the in vitro and in vivo antifungal activity of isoxanthohumol, an isoprene flavonoid from H. lupulus, against Botrytis cinerea; and (3) to explore the antifungal mechanism of isoxanthohumol on B. cinerea. The present data revealed that the ethanolic extract of H. lupulus exhibited moderate antifungal activity against the five tested phytopathogenic fungi in vitro, and isoxanthohumol showed highly significant antifungal activity against B. cinerea, with an EC50 value of 4.32 µg/mL. Meanwhile, it exhibited moderate to excellent protective and curative efficacies in vivo. The results of morphologic observation, RNA-seq, and physiological indicators revealed that the antifungal mechanism of isoxanthohumol is mainly related to metabolism; it affected the carbohydrate metabolic process, destroyed the tricarboxylic acid (TCA) cycle, and hindered the generation of ATP by inhibiting respiration. Further studies indicated that isoxanthohumol caused membrane lipid peroxidation, thus accelerating the death of B. cinerea. This study demonstrates that isoxanthohumol can be used as a potential botanical fungicide for the management of phytopathogenic fungi.