Clonal aggregation of fluconazole-resistant Candida tropicalis isolated from sterile body fluid specimens from patients in Hefei, China.

Candida tropicalis, a human conditionally pathogenic yeast, is distributed globally, especially in Asia-Pacific. The increasing morbidity and azole resistance of C. tropicalis have made clinical treatment difficult. The correlation between clonality and antifungal susceptibility of clinical C. tropicalis isolates has been reported. To study the putative correlation in C. tropicalis isolated from normally sterile body fluid specimens and explore the distinct clonal complex (CC) in Hefei, 256 clinical C. tropicalis isolates were collected from four teaching hospitals during 2016-2019, of which 30 were fluconazole-resistant (FR). Genetic profiles of 63 isolates, including 30 FR isolates and 33 fluconazole-susceptible (FS) isolates, were characterized using multilocus sequence typing (MLST). Phylogenetic analysis of the data was conducted using UPGMA (unweighted pair group method with arithmetic averages) and the minimum spanning tree algorithm. MLST clonal complexes (CCs) were analyzed using the goeBURST package. Among 35 differentiated diploid sequence types (DSTs), 16 DSTs and 1 genotype were identified as novel. A total of 35 DSTs were assigned to five major CCs based on goeBURST analysis. CC1 (containing DST376, 505, 507, 1221, 1222, 1223, 1226, and 1229) accounted for 86.7% (26/30) of the FR isolates. However, the genetic relationships among the FS isolates were relatively decentralized. The local FR CC1 belongs to a large fluconazole non-susceptible CC8 in global isolates, of which the putative founder genotype was DST225. The putative correlation between MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Hefei showed that DSTs are closely related to FR clones.

A local prevalent FR CC1, accounted for 86.7% of the FR isolates in Hefei, China, which showed that fluconazole resistance is closely related to the genetic background, a finding of great value to local medical treatment and possible reasons for the increase in azole resistance of Candida tropicalis.

Understanding etiology of community-acquired central nervous system infections using metagenomic next-generation sequencing.

Background: Community-acquired central nervous system infections (CA-CNS infections) have the characteristics of acute onset and rapid progression, and are associated with high levels of morbidity and mortality worldwide. However, there have been only limited studies on the etiology of this infections. Here, metagenomic next-generation sequencing (mNGS), a comprehensive diagnosis method, facilitated us to better understand the etiology of CA-CNS infections. Methods: We conducted a single-center retrospective study between September 2018 and July 2021 in which 606 cerebrospinal fluid (CSF) samples were collected from suspected CNS infectious patients for mNGS testing, and all positive samples were included in this analysis. Results: After the exclusion criteria, a total of 131 mNGS-positive samples were finally enrolled. Bacterial, viral, fungal, parasitic, specific pathogen and mixed infections were accounted for 32.82% (43/131), 13.74% (18/131), 0.76% (1/131), 2.29% (3/131) and 6.87% (9/131), respectively. A total of 41 different pathogens were identified, including 16 bacteria, 12 viruses, 10 fungi, and 1 parasite and 3 specific pathogens. The most frequent infecting pathogens are Epstein-Barr virus (n = 14), Herpes simplex virus 1 (n = 14), Mycobacterium tuberculosis (n = 13), Streptococcus pneumoniae (n = 13), and Cryptococcus neoformans (n = 8). Some difficult-to-diagnose pathogen infections were also detected by mNGS, such as Streptococcus suis, Pseudorabies virus, Bunyavirus, Orientia tsutsugamushi and Toxoplasma gondii. Conclusion: In this study, mNGS identified a wide variety of pathogens of CA-CNS infections and many of which could not be detected by conventional methods. Our data provide a better understanding of the etiology of CA-CNS infections and show that mNGS represents a comparative screening of CSF in an unbiased manner for a broad range of human pathogens.

Fabrication of Metallic Superhydrophobic Surfaces with Tunable Condensate Self-Removal Capability and Excellent Anti-Frosting Performance.

Laser fabrication of metallic superhydrophobic surfaces (SHSs) for anti-frosting has recently attracted considerable attention. Effective anti-frosting SHSs require the efficient removal of condensed microdroplets through self-propelled droplet jumping, which is strongly influenced by the surface morphology. However, detailed analyses of the condensate self-removal capability of laser-structured surfaces are limited, and guidelines for laser processing parameter control for fabricating rationally structured SHSs for anti-frosting have not yet been established. Herein, a series of nanostructured copper-zinc alloy SHSs are facilely constructed through ultrafast laser processing. The surface morphology can be properly tuned by adjusting the laser processing parameters. The relationship between the surface morphologies and condensate self-removal capability is investigated, and a guideline for laser processing parameterization for fabricating optimal anti-frosting SHSs is established. After 120 min of the frosting test, the optimized surface exhibits less than 70% frost coverage because the remarkably enhanced condensate self-removal capability reduces the water accumulation amount and frost propagation speed (<1 µm/s). Additionally, the material adaptability of the proposed technique is validated by extending this methodology to other metals and metal alloys. This study provides valuable and instructive insights into the design and optimization of metallic anti-frosting SHSs by ultrafast laser processing.

Erratum: LukS-PV Inhibits Hepatocellular Carcinoma Progression by Downregulating HDAC2 Expression.

[This corrects the article DOI: 10.1016/j.omto.2020.05.006.].

LukS-PV Inhibits Hepatocellular Carcinoma Cells Migration via the TNNC1/PI3K/AKT Axis.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. LukS-PV is the S component of Panton-Valentine leucocidin (PVL), a toxin secreted by Staphylococcus aureus. We aimed to investigate the role of LukS-PV in HCC cell migration and the specific molecular mechanism involved. METHODS: We used scratch assays to detect the mobility of liver cancer cells treated with LukS-PV. Quantitative real-time PCR and Western blot analysis were performed to detect the expression levels of related genes. RNA sequencing and quantitative proteomics sequencing were used to assess the transcriptional and proteomic alterations of target genes. RNA sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) pathway analyses revealed the downstream signaling pathway targets of LukS-PV. RESULTS: Our results demonstrated that LukS-PV could inhibit HCC cell migration in a concentration-dependent manner. LukS-PV could also downregulate the expression of TNNC1, which was highly expressed in HCC cells. Additionally, the study showed that LukS-PV inhibited HCC cell migration by downregulating TNNC1. Further studies showed that LukS-PV inhibited the phosphorylation of PI3K/AKT pathway by targeting TNNC1, thereby inhibiting HCC cell migration. CONCLUSION: Our study demonstrated that LukS-PV has an inhibitory role in the migration of liver cancer cells through the TNNC1/PI3K/AKT axis.

LukS-PV Inhibits Hepatocellular Carcinoma Progression by Downregulating HDAC2 Expression.

Hepatocellular carcinoma (HCC) is a common malignant tumor. LukS-PV is the S component of Panton-Valetine leukocidin (PVL), which is secreted by Staphylococcus aureus. This study investigated the effects of LukS-PV on the proliferation, apoptosis, and cell-cycle progression of HCC cells and the mechanisms of its activity. The HCC cells were treated with different LukS-PV concentrations in vitro. Cell Counting Kit-8 and 5-Ethynyl-2'-deoxyuridine (EdU) assays were used to study cell proliferation. Flow cytometry was used to measure apoptosis and cell-cycle progression. Quantitative reverse transcriptase PCR and western blot assays were used to determine mRNA and protein expression levels. Xenograft experiments were performed to determine the in vivo antitumor effect of LukS-PV. Immunostaining was performed to analyze Ki-67 and HDAC2 (histone deacetylase 2) expression. Our results showed that LukS-PV inhibited cell proliferation and induced apoptosis in a concentration-dependent manner in HCC cell lines. LukS-PV also can induce cell-cycle arrest. Moreover, we discovered that LukS-PV attenuated HDAC2 expression and upregulated PTEN; phosphorylated AKT was also reduced. Further studies demonstrated that LukS-PV treatment significantly reduced tumor growth in nude mice and suppressed Ki-67 and HDAC2 levels. Our data revealed a vital role of LukS-PV in suppressing HCC progression by downregulating HDAC2 and upregulating PTEN.

Identification of Hub Genes and Analysis of Prognostic Values in Hepatocellular Carcinoma by Bioinformatics Analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most frequent cancers in the world. In this study, differentially expressed genes (DEGs) between tumor tissues and normal tissues were identified using the comprehensive analysis method in bioinformatics. MATERIALS AND METHODS: We downloaded 3 mRNA expression profiles from the Gene Expression Omnibus database to identify DEGs between tumor tissues and adjacent normal tissues. The Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analysis, protein-protein interaction network was performed to understand the function of DEGs. OncoLnc, which was linked to The Cancer Genome Atlas survival data, was used to investigate the prognostic values of hub genes. The expression of selected hub genes was validated by the quantitative real-time polymerase chain reaction. RESULTS: A total of 235 DEGs, consisting of 36 upregulated and 199 downregulated genes, were identified between tumor tissue and normal tissue. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis results showed the upregulated DEGs to be significantly enriched in cell division, mid-body, ATP binding and oocyte meiosis pathways. The downregulated DEGs were mainly involved in epoxygenase P450 pathway, extracellular region, oxidoreductase activity and metabolic pathways. Ten hub genes, including Aurora kinase A, Cell division cycle 20, formiminotransferase cyclodeaminase, UBE2C, Cyclin B2, pituitary tumor-transforming gene 1, CDKN3, CKS1B, Topoisomerase-II alpha and KIF20A, were identified as the key genes in HCC. Survival analysis found the expression of hub genes to be significantly correlated with the survival of patients with HCC. CONCLUSIONS: The present study identified hub genes and pathways in HCC that may be potential targets for diagnosis, treatment and prognostic prediction.

Quantitative proteomic analysis reveals that Luks-PV exerts antitumor activity by regulating the key proteins and metabolic pathways in HepG2 cells.

Hepatocellular carcinoma (HCC) is a complicated and poor prognosis cancer, necessitating the development of a potential treatment strategy. In this study, we initially revealed that LukS-PV belonged to leukocidin family performs an anti-HCC action. Then, we used liquid chromatography-mass spectrometry (LC/MS) to compare protein expression profiles of the LukS-PV-treated human HCC cell lines HepG2 and the control cells. GO annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis were carried out of differential expression followed by protein-protein interactome, to explore the underlying cancer suppressor mechanisms of LukS-PV for human HCC. A total of 88 upregulated proteins and 46 downregulated proteins were identified. The top 10 proteins identified by the MCC method are FN1, APP, TIMP1, nucleobindin-1, GOLM1, APLP2, CYR61, CD63, ENG, and CD9. Our observation on protein expression indicated that LukS-PV produces a signature affecting central carbon metabolism in cancer, galactose metabolism, and fructose and mannose metabolism pathways. The results give a functional effects and molecular mechanism insight, following LukS-PV treatment.

LukS-PV induces cell cycle arrest and apoptosis through p38/ERK MAPK signaling pathway in NSCLC cells.

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents.

LukS-PV induces apoptosis in acute myeloid leukemia cells mediated by C5a receptor.

LukS-PV is one of the two components of Panton-Valentine leucocidin (PVL). Our previous study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) THP-1 and HL-60 cells. C5aR (C5a receptor) is the receptor for PVL, but whether C5aR plays a key role in LukS-PV induced apoptosis is unclear. The aim of this study was to establish whether C5aR plays a physiological role in apoptosis of leukemia cells induced by LukS-PV. We investigated the role of C5aR in leukemia cell apoptosis induced by LukS-PV by pretreatment of THP-1 and HL-60 cells with C5aR antagonist and transfection to knockdown C5aR in THP-1 cells or overexpress C5aR in Jurkat cells before treatment with LukS-PV. Cell apoptosis was analyzed by staining with Annexin V/propidium iodide or Annexin V-PE/7-AAD. Mitochondrial membrane potential (MMP) was determined using JC-1 dye. The expression of apoptosis-associated genes and proteins was identified by qRT-polymerase chain reaction and Western blotting analysis, respectively. As the C5aR antagonist concentration increased, the rate of apoptosis induced by LukS-PV decreased, the MMP increased, and expression of pro-apoptotic Bax and Bak genes and proteins was downregulated while that of anti-apoptotic Bcl-2 and Bcl-x genes and proteins was upregulated. Knockdown of C5aR also decreased LukS-PV-induced THP-1 cell apoptosis. LukS-PV did not induce apoptosis of Jurkat cells, which have no endogenous C5aR expression; however, LukS-PV did induce apoptosis in Jurkat cells after overexpression of C5aR. Correspondingly, the MMP decreased and Bax and Bak were upregulated while Bcl-2 and Bcl-x were downregulated. LukS-PV can induce apoptosis in AML cells by targeting C5aR. C5aR may be a potential therapeutic target for AML and LukS-PV is a candidate targeted drug for the treatment of AML.

LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2.

BACKGROUND/AIMS: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. METHODS: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. RESULTS: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. CONCLUSIONS: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

Systematic review and meta-analysis of mortality of patients infected with carbapenem-resistant Klebsiella pneumoniae.

PURPOSE: Carbapenem resistant K. pneumoniae (CRKP) has aroused widespread attention owing to its very limited therapeutic options, and this strain has increased rapidly in recent years. Although it is accepted that drug resistance is associated with increased mortality in general, but some other studies found no such relationship. To estimate mortality of patients infected with CRKP in general and analyze factors for mortality of this infection, thus, we conducted this systematic review and meta-analysis. METHODS: A systematic literature review of relevant studies published until December 2015 was conducted. We selected and assessed articles reporting mortality of patients infected with CRKP. RESULTS: Pooled mortality was 42.14% among 2462 patients infected with CRKP versus 21.16% in those infected with carbapenem-susceptible K. pneumoniae (CSKP). The mortality of patients with bloodstream infection (BSI) or urinary tract infection was 54.30 and 13.52%, respectively, and 48.9 and 43.13% in patients admitted to the intensive care unit (ICU) or who underwent solid organ transplantation (SOT). Mortality was 47.66% in patients infected with K. pneumoniae carbapenemase-producing K. pneumoniae and 46.71% in those infected with VIM-producing K. pneumoniae. Geographically, mortality reported in studies from North America, South America, Europe, and Asia was 33.24, 46.71, 50.06, and 44.82%, respectively. CONCLUSIONS: Our study suggests that patients infected with CRKP have higher mortality than those infected with CSKP, especially in association with BSI, ICU admission, or SOT. We also considered that patients' survival has a close relationship with their physical condition. Our results imply that attention should be paid to CRKP infection, and that strict infection control measures and new antibiotics are required to protect against CRKP infection.

VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA.

Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.