Caspase-1-Inhibitor AC-YVAD-CMK Inhibits Pyroptosis and Ameliorates Acute Kidney Injury in a Model of Sepsis.

OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.

Synergistic Potential of Antimicrobial Combinations Against Methicillin-Resistant Staphylococcus aureus.

The chemotherapeutic options for methicillin-resistant Staphylococcus aureus (MRSA) infections are limited. Due to the multiple resistant MRSA, therapeutic failure has occurred frequently, even using antibiotics belonging to different categories in clinical scenarios, very recently. This study aimed to investigate the interactions between 11 antibiotics representing different mechanisms of action against MRSA strains and provide therapeutic strategies for clinical infections. Susceptibilities for MRSA strains were determined by broth microdilution or agar dilution according to CLSI guideline. By grouping with each other, a total of 55 combinations were evaluated. The potential synergism was detected through drug interaction assays and further investigated for time-killing curves and an in vivo neutropenic mouse infection model. A total of six combinations (vancomycin with rifampicin, vancomycin with oxacillin, levofloxacin with oxacillin, gentamycin with oxacillin, clindamycin with oxacillin, and clindamycin with levofloxacin) showed synergistic activity against the MRSA ATCC 43300 strain. However, antibacterial activity against clinical isolate #161402 was only observed when vancomycin combined with oxacillin or rifampicin in time-killing assays. Next, therapeutic effectiveness of vancomycin/oxacillin and vancomycin/rifampicin was verified by an in vivo mouse infection model inoculated with #161402. Further investigations on antimicrobial synergism of vancomycin plus oxacillin and vancomycin plus rifampicin against 113 wild-type MRSA strains were evidenced by combined antibiotic MICs and bacterial growth inhibition and in vitro dynamic killing profiles. In summary, vancomycin/rifampicin and vancomycin/oxacillin are the most potential combinations for clinical MRSA infection upon both in vitro and in vivo tests. Other synergetic combinations of levofloxacin/oxacillin, gentamycin/oxacillin, clindamycin/oxacillin, and clindamycin/fosfomycin are also selected but may need more assessment for further application.

Contralateral C7 transfer combined with acellular nerve allografts seeded with differentiated adipose stem cells for repairing upper brachial plexus injury in rats.

Nerve grafting has always been necessary when the contralateral C7 nerve root is transferred to treat brachial plexus injury. Acellular nerve allograft is a promising alternative for the treatment of nerve defects, and results were improved by grafts laden with differentiated adipose stem cells. However, use of these tissue-engineered nerve grafts has not been reported for the treatment of brachial plexus injury. The aim of the present study was to evaluate the outcome of acellular nerve allografts seeded with differentiated adipose stem cells to improve nerve regeneration in a rat model in which the contralateral C7 nerve was transferred to repair an upper brachial plexus injury. Differentiated adipose stem cells were obtained from Sprague-Dawley rats and transdifferentiated into a Schwann cell-like phenotype. Acellular nerve allografts were prepared from 15-mm bilateral sections of rat sciatic nerves. Rats were randomly divided into three groups: acellular nerve allograft, acellular nerve allograft + differentiated adipose stem cells, and autograft. The upper brachial plexus injury model was established by traction applied away from the intervertebral foramen with micro-hemostat forceps. Acellular nerve allografts with or without seeded cells were used to bridge the gap between the contralateral C7 nerve root and C5-6 nerve. Histological staining, electrophysiology, and neurological function tests were used to evaluate the effect of nerve repair 16 weeks after surgery. Results showed that the onset of discernible functional recovery occurred earlier in the autograft group first, followed by the acellular nerve allograft + differentiated adipose stem cells group, and then the acellular nerve allograft group; moreover, there was a significant difference between autograft and acellular nerve allograft groups. Compared with the acellular nerve allograft group, compound muscle action potential, motor conduction velocity, positivity for neurofilament and S100, diameter of regenerating axons, myelin sheath thickness, and density of myelinated fibers were remarkably increased in autograft and acellular nerve allograft + differentiated adipose stem cells groups. These findings confirm that acellular nerve allografts seeded with differentiated adipose stem cells effectively promoted nerve repair after brachial plexus injuries, and the effect was better than that of acellular nerve repair alone. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University of China (approval No. 2016-150) in June 2016.

Combination Therapy Strategies Against Multiple-Resistant Streptococcus Suis.

Streptococcus suis is a major swine pathogen, an emerging zoonotic agent responsible for meningitis, endocarditis and septicaemia followed by deafness in humans. The development of antimicrobial resistance in S. suis increases the risk for therapeutic failure in both animals and humans. In this study, we report the synergism of combination therapy against multi-resistant S. suis isolates from swine. Twelve antibiotic profiles were determined against 11 S. suis strains. To investigate their synergistic/antagonistic activity, checkerboard assay was performed for all the possible combinations. In-vitro killing curves and in-vivo treatment trials were used to confirm the synergistic activity of special combinations against S. suis dominant clones. In this study, 11 S. suis isolates were highly resistant to erythromycin, clindamycin, trimethoprim/sulfamethoxazole, and tetracycline with ratios of 80-100%, and the resistance percentages to enrofloxacin, florfenicol, and spectinomycin were ~50%. The checkerboard data identified two combination regimens, ampicillin plus apramycin and tiamulin plus spectinomycin which gave the greatest level of synergism against the S. suis strains. In-vitro kill-curves showed a bacterial reduction of over 3-logCFU with the use of combination treatments, whilst the application of mono-therapies achieve less than a 2-logCFU cell killing. In-vivo models confirm that administration of these two combinations significantly reduced the number of bacterial cells after 24 h of treatment. In conclusions, the combinations of ampicillin plus apramycin and tiamulin plus spectinomycin showed the greatest synergism and may be potential strategies for treatment of multi-resistant S. suis in animal.

Optimization of an Activity Assay of Coprinus Cinereus Peroxidase

ABSTRACT To seek a simple, rapid and sensitive Coprinus cinereus Peroxidase (CIP) activity assay, a convenient one-factor-at-a-time (OFAT) method and a response surface methodology (RSM) were used. The recombinant CIP expressed in Pichia pastoris was purified with the Ni-NTA spin column. Based on the results of catalytic efficiency (kcat/Km) analysis, 2,2'-azinobis (ethylbenzthiazoline -6-sulfonate) (ABTS) was selected as the optimal enzyme substrate. Results of the OFAT method showed that enzymatic reaction performed in 0.1 mol/L sodium acetate (pH 5.0) buffer in a 200-µl reaction mixture containing 0.5 mmol/L ABTS, 10 mmol/L hydrogen peroxide (H2O2), 49.7 ng CIP at 25°C gave an average CIP activity of 88 U/mL. The ABTS and H2O2 concentrations were then further optimized to improve the sensitivity of the assay. To do that, RSM was conducted through central composite design, and a reduced quadratic model with good fit regression equation was generated. ANOVA analysis of this model indicated that the concentrations of ABTS and H2O2 and their interaction had significant impact on the assay sensitivity. The optimal reaction mixture was determined to include an initial ABTS concentration of 0.82 mmol/L 49.7 ng CIP and 16.36 mmol/L H2O2, and the activity under this condition was determined to be 138.89 U/mL.

Efficacy of Cefquinome against Escherichia coli Environmental Mastitis Assessed by Pharmacokinetic and Pharmacodynamic Integration in Lactating Mouse Model.

This work investigates the pharmacodynamic effectiveness of cefquinome against environmental Escherichia coli mastitis infection, following an intramammary administration. We established the pharmacokinetic and pharmacodynamic (PK/PD) model in lactating mice. The PK/PD parameters were identified to achieve an antibacterial efficacy as indicated by PD activity, cytokine expression and PK/PD simulation. From our findings, given an 200 µg/gland dose once daily can achieve a considerable therapeutic effectiveness in experimental circumstance.

Clonal spread of mcr-1 in PMQR-carrying ST34 Salmonella isolates from animals in China.

Since initial identification in China, the widespread geographical occurrence of plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae has been of great concern. In this study, a total of 22 Salmonella enterica were resistant to colistin, while only five isolates which belonged to ST34 Salmonella enterica serovar Typhimurium (S. Typhimurium) were mcr-1 positive. Four of them shared nearly identical PFGE type, although they were from different host species and diverse geographical locations. All the mcr-1-positive S. Typhimurium exhibited multi-resistant phenotypes including ampicillin, streptomycin, gentamicin, florfenicol, nalidixic acid, tetracycline, trimethoprim-sulfamethox, in addition to colistin. The oqxAB and aac(6')-Ib-cr genes were present alone or in combination in four (80.0%) and five (100%) isolates, respectively. The mcr-1 gene was located on a transferable IncI2 plasmid in the four genetically related strains. In the other one strain, mcr-1 was located on an approximately 190 kb IncHI2 plasmid. In conclusion, we report five mcr-1-positive S. Typhimurium/ST34 isolates. Both clonal expansion and horizontal transmission of IncI2-type plasmids were involved in the spread of the mcr-1 gene in Salmonella enterica from food-producing animals in China. There is a great need to monitor the potential dissemination of the mcr-1 gene.