Dual inhibitors of ASK1 and PDK1 kinases: Design, synthesis, molecular docking and mechanism studies of N-benzyl pyridine-2-one containing derivatives as anti-fibrotic agents.

Utilizing fragment-based hybrid designing strategies, 24 N-benzyl pyridine-2-one containing derivatives were synthesized by successfully incorporating 6-(4H-1,2,4-triazol-3-yl) pyridin-2-amine of scaffold of ASK1 inhibitor (GS-444217). These newly synthesized compounds were screened in cell-free ASK1 and PDK1 kinase and cellular vitality assays. Among all compounds tested, both 21c and 21d displayed single digit potency of 9.13, 1.73 nM in inhibiting ASK1, and exhibited excellent enzyme inhibitory activity against PDK1 (the inhibition rates at 10 µM were 13.63% and 23.80%, respectively). Specifically, both compounds inhibited the TGF-ß1 induced fibrotic response and blocked the up-regulated protein expression levels of ASK1-p38/JNK signaling pathways and possessed the potency in reducing PDK1/Akt phosphorylation. The results herein showed the potential lead characteristics of 21c or 21d as dual inhibitors ASK1/PDK1 kinases.

Antifungal susceptibility profiles and drug resistance mechanisms of clinical Candida duobushaemulonii isolates from China.

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 µg/ml. One isolate showed a high MIC value of ≥64 µg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment.

Analysis of hypervirulent Klebsiella pneumoniae and classic Klebsiella pneumoniae infections in a Chinese hospital.

AIMS: To evaluate the clinical and genetic virulence characteristics of critically ill patients with hypervirulent Klebsiella pneumoniae (hvKP) and classic KP (cKP) infection. METHODS AND RESULTS: The patients included in this retrospective study (n = 225) were grouped according to their hvKP (n = 114) or cKP (n = 111) status, and their clinical characteristics were analysed and compared. Cox multivariate analysis was conducted to determine the risk factors for hvKP infection. Length of hospital stay, length of intensive care unit stay, duration of mechanical ventilation and 28-day survival rate were similar between the groups. However, the incidence of septic shock was higher in the hvKP group (16.7%) than in the cKP group (8.1%). CONCLUSIONS: There was a high rate of hvKP infection in this population. Compared to patients with cKP infection, those with hvKP infection showed a higher probability of having septic shock; nevertheless, survival and length of hospital stay were similar between the groups. Risk factors for hvKP infection included hospital-acquired infection and renal insufficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents relevant information on the characteristics of hvKP infection in a Chinese population, and this promotes early diagnosis and supports the view that the prevalence of hvKP is high in China.

A circular RNA derived from MMP9 facilitates oral squamous cell carcinoma metastasis through regulation of MMP9 mRNA stability.

Emerging evidence demonstrates that dysregulation of circular RNA is linked to tumorigenesis and aggressive progression. However, its role in oral squamous cell carcinoma remains largely uncharacterized. In this study, we identified a novel metastasis-associated circular RNA, circular matrix metalloproteinase 9 (hsa_circ_0001162, a circular RNA derived from matrix metalloproteinase 9), which was remarkably upregulated in oral squamous cell carcinoma and positively correlated with matrix metalloproteinase 9 expression. Patients with high circular matrix metalloproteinase 9 expression were prone to lymph node metastasis and an advanced TNM stage. Importantly, circular matrix metalloproteinase 9 was identified as an efficacious diagnostic and prognostic biomarker for oral squamous cell carcinoma patients. Functional experiments showed that depletion of circular matrix metalloproteinase 9 weakened the migratory and invasive capabilities of oral squamous cell carcinoma cells in vitro as well as inhibited lung metastasis in vivo. Regarding the mechanism, circular matrix metalloproteinase 9 could simultaneously interact with AUF1 and miR-149 to block the inhibitory effect of AUF1 and miR-149 on matrix metalloproteinase 9 3'-untranslated region, resulting in enhanced matrix metalloproteinase 9 messenger RNA stability, thereby facilitating oral squamous cell carcinoma metastasis. Collectively, our data indicate that circular matrix metalloproteinase 9 acts as a metastasis-promoting gene in oral squamous cell carcinoma through regulating the messenger RNA stability of its parental gene. Therapeutic targeting of circular matrix metalloproteinase 9 may be a promising treatment intervention for metastatic oral squamous cell carcinoma patients.

Clinical Situations of Bacteriology and Prognosis in Patients with Urosepsis.

BACKGROUND: Urosepsis and septic shock are a critical situation leading to a mortality rate up to 30% in patients with obstructive diseases of the urinary tract. AIM: To analyze the bacterial distribution and drug resistance of pathogenic bacteria in patients with urosepsis and to provide a basis for the rational application of antibacterial drugs in clinical practice. METHODS: A retrospective analysis of 94 hospitalized patients with urosepsis for 6 years was performed. The strain composition, resistance characteristics, and the antibiogram of common bacteria from positive blood and midstream urine culture were analyzed. RESULTS: A total of 87 strains were isolated, including 65 strains (74.71%) of Gram-negative bacilli, 14 strains (16.09%) of Gram-positive cocci, and 8 strains (9.20%) of fungi. The Gram-negative bacilli included 42 strains of Escherichia coli (E. coli) (64.62%), among which 34 strains (80.95%) were producing ESBLs, and 14 strains (21.84%) of Klebsiella pneumoniae (K. pneumoniae), among which nine strains (64.29%) were producing ESBLs. The most common pathogenic bacteria, ESBL+ E. coli and K. pneumoniae strains, showed sensitivity towards imipenem, ertapenem, piperacillin/tazobactam, amikacin, and cefotetan, but were highly resistant to quinolones. The cure rate of urosepsis was 88.30%, and the susceptibility rate of septic shock was 45.47%. SIGNIFICANCE: Gram-negative bacterial infections are the main cause of urosepsis. The mild patient group showed more E. coli (ESBL-) infections, and the number of ESBL producing E. coli isolated from the mild group showed higher drug resistance rates for aztreonam and levofloxacin compared with isolates from the severe group.

Co-outbreak of multidrug resistance and a novel ST3006 Klebsiella pneumoniae in a neonatal intensive care unit: A retrospective study.

The outbreak of carbapenem-resistant Klebsiella pneumoniae is a serious public health problem, especially in the neonatal intensive care unit (NICU).Fifteen K. pneumoniae strains were isolated from 7 neonates during June 3 to 28, 2017 in an NICU. Antimicrobial susceptibility was determined by the Vitek 2 system and microbroth dilution method. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used to analyze the genetic relatedness of the isolates. Whole-genome sequencing and gene function analysis were performed to investigate pathogenicity and drug resistance and screen genomic islands.Three clones of K. pneumoniae were identified from 7 neonates: 7 strains of ST37, 7 of novel ST3006, and 1 of ST1224. Gene sequencing showed that the kpn1343 (ST37) strain harbored 12 resistance genes (OXA-33, TEM-1, SHV-11, AAC (6')-IId, AAC (3)-IIa, AAC (6')-Ib-cr, catB3, arr-3, sul1, oqxB, oqxA, CRP, and catB3) and included 15 genomic islands and 205 reduced virulence genes. The kpn1344 (ST3006) strain harbored 4 antibiotic-resistant genes (TEM-1, CTX-M-3, vgaC, and CRP) and included 19 genomic islands and 209 reduced virulence genes. MLST and PFGE showed that 15 strains of K. pneumoniae were divided into 3 groups with a high level of homology. ST1224 (kpn1362) was isolated on June 28, 2017, which was 10 days after the last isolate (kpn1359, June 18, 2017); thus, we speculated that ST1224 was not the clone that caused the outbreak.This co-outbreak of K. pneumoniae involved 2 clones: ST37 and ST3006. ST37 carried the multidrug-resistant genes, such as OXA-33, TEM-1, and SHV-11, and ST3006 was a novel K. pneumoniae ST typing. Whole-genome sequencing may be an effective method for screening bacterial-resistant genes and their functions.

CusS-CusR Two-Component System Mediates Tigecycline Resistance in Carbapenem-Resistant Klebsiella pneumoniae.

BACKGROUND: The increase in carbapenem-resistant Klebsiella pneumoniae (CRKP), especially the emergence of tigecycline-resistant K. pneumoniae (KP), is a serious public health concern. However, the underlying mechanism of tigecycline resistance is unclear. In this study, we evaluated the role of the CusS-CusR two-component system (TCS), which is associated with copper/silver resistance, in tigecycline resistance in CRKP. METHODS: Following the in vitro evolution of tigecycline-resistant KP, the minimum inhibitory concentrations of tigecycline were determined using the micro-broth dilution method. RNA sequencing and data analysis were performed to identify differentially expressed genes. Quantitative PCR (qPCR) was performed to verify the genes of interest. Genes associated with tigecycline resistance, such as ramR, tex (T), and tet (A), were detected by PCR, and then mutants were confirmed by sequencing. Additionally, the efflux pump-associated genes soxS, oqxA, oqxB, acrE, and acrF were also analyzed by qPCR. CusR was deleted and complemented by the suicide vector pKO3-Km plasmid and pGEM-T-easy plasmid, respectively. RESULTS: Nine strains of KP were evaluated in our study. Strains A2 and A3 were evolved from A1, B2, and B3 were evolved from B1, and C2 and C3 were evolved from C1. The tigecycline minimum inhibitory concentration for A1, B1, and C1 was 0.5 µg/mL; that for A2, B2, and C3 was 16.0 µg/mL; and that for A3, B3, and C3 was 32.0 µg/mL. RNA-sequencing and qPCR confirmed that the differentially expressed genes cusE, cusS, cusR, cusC, cusF, cusB, and cusA showed higher expression in C2 and C3 than in C1. Genes related to the efflux pump AcrAB-TolC showed higher expression in B2 and B3 than in B1. No mutants of ramR, tex (T), or tet (A) were detected. SoxS, oqxA, oqxB, acrE, and acrF did not show increased expression in any group. After deletion and complementation of cusR among C3, the MIC of tigecycline decreased to 4 µg/mL, and then recovered to 32 µg/mL. The expression of cusFBCA, correspondingly decreased and increased significantly. CONCLUSION: In addition to its primary function in resistance to copper/silver, the CusS-CusR two-component system is associated with CRKP resistance to tigecycline.

Development of a bead-based suspension array for the detection of pathogens in acute respiratory tract infections.

We developed a high-throughput bead-based suspension array for simultaneous detection of 20 respiratory tract pathogens in clinical specimens. Pathogen-specific genes were amplified and hybridized to probes coupled to carboxyl-encoded microspheres. Fluorescence intensities generated via the binding of phycoerythrin-conjugated streptavidin with biotin-labeled targets were measured by the Luminex 100 bead-based suspension array system. The bead-based suspension array detected bacteria in a significantly higher number of samples compared to the conventional culture. There was no significant difference in the detection rate of atypical pathogensatypical pathogens or viruses between the bead-based suspension array and real-time PCR. This technology can play a significant role in screening patients with pneumonia.

Comparison of Talaromyces marneffei Infection in Human Immunodeficiency Virus-positive and Human Immunodeficiency Virus-negative Patients from Fujian, China.

BACKGROUND: Talaromyces (Penicillium) marneffei (TM) is an emerging dimorphic human pathogenic fungus that is endemic to Southeast Asia. TM mostly occurs as an opportunistic infection in patients with human immunodeficiency virus (HIV). The objective of this study was to compare the clinical and laboratory parameters of patients with TM infections who were HIV-positive and HIV-negative and to assess therapies and outcomes. METHODS: This was a retrospective analysis of 26 patients diagnosed with disseminated TM infection from September 2005 to April 2014 at Fujian Provincial Hospital, China. RESULTS: Patients with TM infection tend to present with fever, weight loss, and anemia. The time from symptom onset to confirmed diagnosis was greater for HIV-negative patients (n = 7; median: 60 days, range: 14-365 days) than for HIV-positive patients (n = 19; median: 30 days, range: 3-90 days, Mann-Whitney U = 31.50, P= 0.041). HIV-negative patients were more likely to have dyspnea (57.1% vs. 5.3%, χ2 = 8.86, P= 0.010), low neutrophil count (Mann-Whitney U = 27.00, P= 0.029), high CD4 count (Mann-Whitney U = 0.00, P= 0.009), and high lymphocyte count (Mann-Whitney U = 21.00, P= 0.009). There were no significant differences in other demographic, clinical, or biochemical characteristics. Among all the patients, 12 HIV-positive patient and 1 HIV-negative patient received amphotericin and fluconazole treatment, 9 of whom improved, 1 died, 2 had kidney damage, 1 had hypokalemia due to exceeded doses. CONCLUSIONS: HIV-negative patients with TM infections tend to have a longer diagnostic interval, a higher percentage of dyspnea, higher levels of CD4 and lymphocytes, and lower neutrophil counts than TM infection in HIV-positive patients. Treatment programs with amphotericin and fluconazole are mostly effective.

Association of Salivary Helicobacter pylori Infection with Oral Diseases: a Cross-sectional Study in a Chinese Population.

OBJECTIVE: The aim of this study was to detect the prevalence of oral H.pylori among adults and to investigate the correlation between H.pylori infection and common oral diseases. STUDY DESIGN: A cross-sectional study was performed among adults Chinese who took their annual oral healthy examination at The First Affiliated Hospital, Zhejiang University School of Medicine, China. RESULTS: The study included 1050 subjects in total and oral H.pylori infection occurred in 60.29% of the subjects. The prevalence rates of oral H.pylori in patients with periodontal diseases (63.42%) and caries (66.91%) were significantly increased than those without oral diseases (54.07%), respectively (P < 0.05), while the difference between subjects with recurrent aphthous stomatitis and controls was not significant. In addition, the differences of positive rates of H.pylori with or without history of gastric ulcer were statistically significant (69.47% vs 58.26%, P<0.05). Presenting with periodontal diseases (OR 1.473;95% CI 1.021 to 2.124), caries (OR 1.717; 1.127 to 2.618), and having history of gastric ulcer (OR 1.631; 1.164 to 2.285) increased the risk of H.pylori infection. CONCLUSIONS: Oral H.pylori infection is common in adult Chinese, which is significantly associated with oral diseases including periodontal diseases and caries.

[The application of loop-mediated isothermal amplification for detection of common pathogenic bacteria in lower respiratory tract infections].

OBJECTIVE: To investigate the spectrum of common pathogenic bacteria of low respiratory tract infection by loop-mediated isothermal amplification (LAMP) of nucleic acid test and to prove the clinical significance of this method. METHODS: A total of 289 qualified sputum samples from patients with lower respiratory tract infections in Fujian Province were detected by LAMP technique, and then the distribution of pathogenic bacteria was analyzed. The positive cases (the patients whose specific bacterial copies in their sputum samples > 1×10(3) copies/ml) were divided into 2 groups according to whether their treatment had covered this pathogen or not. The underlying diseases, duration of anti-bacterial treatment, the hospital days, and the effectiveness of initial treatment and cure rate were compared. RESULTS: The culture method in the 289 patients showed that 44 (15.2%) were positive. Tests by the LAMP method with a bacteria concentration > 1×10(3) copies/ml as cutoff value, showed positive results in 124 patients (43.0%). The lower respiratory tract pathogens included 144 strains of bacteria (77.8%), and 41 strains of atypical pathogens (22.2%). Gram-negative bacteria were the predominant pathogens, such as Pseudomonas aeruginosa, H. influenzae, Klebsiella pneumoniae, and Streptococcus pneumoniae. In 95 cases the initial therapy had covered the pathogens, while in 29 cases the initial therapy had not. The effectiveness of the initial treatment (χ(2) = 31.0, P < 0.01) and the total days of hospital stay (t = -2.083, P = 0.039) in the group whose antibiotics had covered the pathogens were significantly higher than those of the other group. However, there were no significant difference in duration of anti-bacterial treatment (t = -1.073, P = 0.285)and cure rates (χ(2) = 0.6, P = 0.4) between the 2 groups. CONCLUSIONS: LAMP method can detect the nuclear acid of the bacteria in the sputum much more rapidly and sensitively than the routine culture method. LAMP technique may be helpful to know the pathogenic bacteria before treatment, and therefore may improve the choice of initial antibiotic therapy.