Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex polymerase chain reaction (PCR) amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising six previously reported alleles and eight new nonsynonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by coexpressing them with cytochromes P450 oxidoreductase (CYPOR) in Saccharomyces cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was used as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, whereas the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. SIGNIFICANCE STATEMENT: In this study, we established a multiplex polymerase chain reaction (PCR) amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these eight novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Impact of high-power short-duration atrial fibrillation ablation technique on the incidence of silent cerebral embolism: a prospective randomized controlled study.

BACKGROUND: High-power short-duration (HPSD) ablation strategy has emerged as a popular approach for treating atrial fibrillation (AF), with shorter ablation time. The utilized Smart Touch Surround Flow (STSF) catheter, with 56 holes around the electrode, lowers electrode-tissue temperature and thrombus risk. Thus, we conducted this prospective, randomized study to investigate if the HPSD strategy with STSF catheter in AF ablation procedures reduces the silent cerebral embolism (SCE) risk compared to the conventional approach with the Smart Touch (ST) catheter. METHODS: From June 2020 to September 2021, 100 AF patients were randomized 1:1 to the HPSD group using the STSF catheter (power set at 50 W) or the conventional group using the ST catheter (power set at 30 to 35 W). Pulmonary vein isolation was performed in all patients, with additional lesions at operator's discretion. High-resolution cerebral diffusion-weighted magnetic resonance imaging (hDWI) with slice thickness of 1 mm was performed before and 24-72 h after ablation. The incidence of new periprocedural SCE was defined as the primary outcome. Cognitive performance was assessed using the Montreal Cognitive Assessment (MoCA) test. RESULTS: All enrolled AF patients (median age 63, 60% male, 59% paroxysmal AF) underwent successful ablation. Post-procedural hDWI identified 106 lesions in 42 enrolled patients (42%), with 55 lesions in 22 patients (44%) in the HPSD group and 51 lesions in 20 patients (40%) in the conventional group (p = 0.685). No significant differences were observed between two groups regarding the average number of lesions (p = 0.751), maximum lesion diameter (p = 0.405), and total lesion volume per patient (p = 0.669). Persistent AF and CHA2DS2-VASc score were identified as SCE determinants during AF ablation procedure by multivariable regression analysis. No significant differences in MoCA scores were observed between patients with SCE and those without, both immediately post-procedure (p = 0.572) and at the 3-month follow-up (p = 0.743). CONCLUSIONS: Involving a small sample size of 100 AF patients, this study reveals a similar incidence of SCE in AF ablation procedures, comparing the HPSD strategy using the STSF catheter to the conventional approach with the ST catheter. TRIAL REGISTRATION: Clinicaltrials.gov: NCT04408716. AF = Atrial fibrillation, DWI = Diffusion-weighted magnetic resonance imaging, HPSD = High-power short-duration, ST = Smart Touch, STSF = Smart Touch Surround Flow.

A fluorescent probe for specifically measuring the overall thioredoxin and glutaredoxin reducing activity in bacterial cells.

Thioredoxins (Trxs) and glutaredoxins (Grxs) are the two major thiol-dependent reductases, participating in many important cellular events such as defense against oxidative stress, DNA synthesis and repair. Both Trxs and Grxs have diverse disulfide-containing substrates in the cells to exert their activities, with overlapping functions. Specific methods for measuring the intracellular overall activities of Trxs and Grxs are still lacking. Here we find that TRFS-green, a disulfide containing fluorescent probe which was used to detect thioredoxin reductase (TrxR) in mammalian cells, is a substrate of bacterial Trxs and Grxs, but not a substrate of bacterial TrxR and GSH. This property made TRFS-green work as a probe to measure the overall activities of Trxs and Grxs in bacterial cells. Using various E. coli Trx or Grx null mutant strains, the contribution of different Trxs and Grxs to cellular redox regulation has been clarified, judged by the reducibility towards TRFS-green. E. coli Grx2 and Grx3 unexpectedly exhibited higher activity in reducing the disulfide probe than the other redoxins. In addition, the bacterial disulfide reductase activity was detected to be affected in the ofloxacin bactericidal process. These results show that TRFS-green may be a useful tool for investigating bacterial redox regulation and demonstrating the critical role of E. coli Grxs in maintaining the bacterial intracellular redox balance.

Prednisone Ameliorates Atrial Inflammation and Fibrosis in Atrial Tachypacing Dogs.

Atrial inflammation and fibrosis have long been considered culprits in the development of atrial fibrillation (AF). Prior clinical studies showed that corticosteroid therapy is beneficial in patients with AF. Here we sought to determine whether prednisone treatment prevents atrial tachypacing (ATP) induced atrial fibrosis.Dogs were randomized into the sham, ATP, ATP + low-dose prednisone (ALP), and ATP + high-dose prednisone (AHP) groups. After 6 days of recovery from surgery, dogs were subjected to ATP at 400 beats per minute for 4 weeks while being treated with prednisone (15 or 40 mg/day) or a placebo. Pacemakers were not activated in the sham group.Compared with the ATP group, the expression of collagen I, collagen III, α-smooth muscle actin, transforming growth factor-ß1 and connective tissue growth factor were significantly reduced in the ALP and AHP groups. Fluorescence assays showed that reactive oxygen species formation in the right atrium was suppressed in the ALP and AHP groups compared with the ATP group. The protein level of NADPH oxidase 2 was reduced in the ALP and AHP groups' versus ATP group, while NOX4 and NOX5 were unchanged. ATP-induced downregulation of BH4 and eNOS uncoupling in the atria was partially restored in the prednisone-treated groups.Our study demonstrated that atrial fibrosis induced by ATP were suppressed by prednisone. Low-dose prednisone was also effective in suppressing the development of atrial fibrosis.

TGF-ß isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners.

Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.

Thioredoxin reductase as a pharmacological target.

Thioredoxin reductases (TrxRs) belong to the pyridine nucleotide disulfide oxidoreductase family enzymes that reduce thioredoxin (Trx). The couple TrxR and Trx is one of the major antioxidant systems that control the redox homeostasis in cells. The thioredoxin system, comprised of TrxR, Trx and NADPH, exerts its activities via a disulfide-dithiol exchange reaction. Inhibition of TrxR is an important clinical goal in all conditions in which the redox state is perturbed. The present review focuses on the most critical aspects of the cellular functions of TrxRs and their inhibition mechanisms by metal ions or chemicals, through direct targeting of TrxRs or their substrates or protein interactors. To update the involvement of overactivation/dysfunction of TrxRs in various pathological conditions, human diseases associated with TrxRs genes were critically summarized by publicly available genome-wide association study (GWAS) catalogs and literature. The pieces of evidence presented here justify why TrxR is recognized as one of the most critical clinical targets and the growing current interest in developing molecules capable of interfering with the functions of TrxR enzymes.

Dietary and metabolic risk of neuropsychiatric disorders: insights from animal models.

Neuropsychiatric disorders are major causes of the global burden of diseases, frequently co-occurring with multiple co-morbidities, especially obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease and its various risk factors in the metabolic syndrome. While the determining factors of neuropsychiatric disorders are complex, recent studies have shown that there is a strong link between diet, metabolic state and neuropsychiatric disorders, including anxiety and depression. There is no doubt that rodent models are of great value for preclinical research. Therefore, this article focuses on a rodent model of chronic consumption of high-fat diet (HFD), and/or the addition of a certain amount of cholesterol or sugar, meanwhile, summarising the pattern of diet that induces anxiety/depressive-like behaviour and the underlying mechanism. We highlight how dietary and metabolic risk influence neuropsychiatric behaviour in animals. Changes in dietary patterns, especially HFD, can induce anxiety- or depression-like behaviours, which may vary by diet exposure period, sex, age, species and genetic background of the animals used. Furthermore, dietary patterns significantly aggravate anxiety/depression-like behaviour in animal models of neuropsychiatric disorders. The mechanisms by which diet induces anxiety/depressive-like behaviour may involve neuroinflammation, neurotransmitters/neuromodulators, neurotrophins and the gut-brain axis. Future research should be focused on elucidating the mechanism and identifying the contribution of diet and diet-induced metabolic risk to neuropsychiatric disorders, which can form the basis for future clinical dietary intervention strategies for neuropsychiatric disorders.

Depletion of multidrug-resistant uropathogenic Escherichia coli BC1 by ebselen and silver ion.

Ebselen, an organo-selenium compound with well-characterized toxicology and pharmacology, recently exhibited potent antibacterial activity against glutathione (GSH)-negative bacteria by disrupting redox homeostasis. In this paper, we show that ebselen and silver ion in combination exert strong bactericidal activity against multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) BC1, a model MDR GSH-positive bacterium. The mechanisms were found to involve consumption of total intracellular GSH and inhibition of thioredoxin reductase activity, which was highly related to reactive oxygen species up-regulation. Furthermore, the therapeutic efficacy of ebselen and silver ion against UPEC-induced cystitis was assessed in a mouse model. Treatment with ebselen and silver ion significantly reduced bacterial loads, down-regulated the expression levels of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on-site and decreased white/red blood cell counts in mild cystitis model mice, which demonstrated the anti-inflammatory property of these agents. In addition, ebselen and silver ion also exhibited significantly high protective ability (100%) against acute cystitis infections. These results together may lay the foundation for further analysis and development of ebselen and silver ion as antibacterial agents for treatment of MDR UPEC infections.

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia.

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.

Rapid and continuous on-chip loading of trehalose into erythrocytes.

Freeze-drying is a promising approach for the long-term storage of erythrocytes at room temperature. Studies have shown that trehalose loaded into erythrocytes plays an important role in protecting erythrocytes against freeze-drying damage. Due to the impermeability of the erythrocyte membrane to trehalose, many methods have been developed to load trehalose into erythrocytes. However, these methods usually require multistep manual manipulation and long processing time; the adopted protocols are also diverse and not standardized. Thus, we develop an osmotically-based trehalose-loading microdevice (TLM) to rapidly, continuously, and automatically produce erythrocytes with loaded trehalose. In the TLM, trehalose is loaded through the erythrocyte membrane pores induced by hypotonic shock; then, the trehalose-loaded erythrocytes are rinsed to remove hemoglobin molecules and cell fragments, and the extracellular solution is restored to the isotonic state by integrating a rinsing-recovering design. First, the mixing function and the rinsing-recovering function were confirmed using a fluorescent solution. Then, the performance of the TLM was evaluated under various operating conditions with respect to the loading efficiency of trehalose, the hemolysis rate of erythrocytes (ϕ), the recovery rate of hemoglobin in erythrocytes (φ), and the separation efficiency of the TLM. Finally, the preliminary study of the freeze-drying of erythrocytes with loaded trehalose was accomplished using the TLM. The results showed that under the designated operating conditions, the loading efficiency for human erythrocytes reached ~21 mM in ~2 min with a ϕ value of ~17% and a φ value of ~74%. This study provides insights into the design of the on-chip loading of trehalose into erythrocytes and promotes the automation of life science studies on biochips.

Intravascular cells and circulating microparticles induce procoagulant activity via phosphatidylserine exposure in heart failure.

Relatively little information is known about the definitive role of phosphatidylserine (PS) in the hypercoagulability of heart failure (HF). Our objectives were to assess the levels of PS exposure on microparticles (MPs) and blood cells (BCs) in each group of HF patients and to evaluate their procoagulant activity (PCA). HF patients in each NYHA functional class II-IV (II n = 30, III n = 30, IV n = 30) and healthy controls (n = 25) were enrolled in the present study. PS exposure on MPs, BCs was analyzed with flow cytometry. MPs were classified based on their cellular origin: platelets (CD41a+), neutrophils (CD66b+), endothelial cells (CD31+CD41a-), erythrocytes (CD235a+), monocytes (CD14+), T lymphocytes (CD3+), and B lymphocytes (CD19+). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+ BCs and MPs were performed by lactadherin. There was no significant difference in MP cellular origin between healthy and HF subjects. However, the total number of PS+ MPs was significantly increased in HF patients compared with healthy controls. In addition, circulating PS+ BCs cooperated with PS+ MPs to markedly shorten coagulation time and dramatically increase FXa/thrombin generation and fibrin formation in each HF group. Moreover, blockade of exposed PS on BCs and MPs with lactadherin inhibited PCA by approximately 80%. Our results lead us to believe that exposing PS on the injured BCs and MPs played a pivotal role in the hypercoagulability state in HF patients.

The roles of SOCS3 and STAT3 in bacterial infection and inflammatory diseases.

The suppressor of cytokine signalling-3 (SOCS3) is well known as a feedback inhibitor of the Janus kinases-signal transducer and activator of transcription 3 (JAK/STAT3) signalling pathway, which mediates the signal transduction of many cytokines, growth factors and hormones during many cellular processes. The critical role of SOCS3 is manifested by its binding to both the JAK kinase and the cytokine receptor, which can result in the inhibition of STAT3 phosphorylation. STAT3 triggers variety of genes expression in response to cytokine (IL-6 family, IL-10) and growth factor stimulation and thus plays a critical role in many cellular biological processes involved in anti/pro-inflammatory responses, cell growth and cell death. SOCS3 might act directly either by hampering JAK activation or by mediating the ubiquitination and subsequent proteasome degradation of the cytokine/growth factor/hormone receptor. Generally, SOCS3 is a negative regulator for cytokine or hormone signalling. But in some cases, SOCS3 regulates the inflammatory responses positively through inhibiting STAT3. An increasing number of reports showed abnormal expression levels of SOCS3/STAT3 in different myeloid and lymphoid cells as well as in various non-hematopoietic cells, which suggested its involvement in various infection and inflammatory diseases. In this review, we described the role of SOCS3 and STAT3 expression in different cell populations in regulating the outcomes of infection and inflammatory diseases.

Simulation of the osmosis-based drug encapsulation in erythrocytes.

Drug-loaded erythrocytes have been proposed for the treatment of disease. A common way to load drugs into erythrocytes is to apply osmotic shock. Currently, osmosis-based drug encapsulation is studied mainly experimentally, whereas a related theoretical model is still incomplete. In this study, a set of equations is developed to simulate the osmosis-based drug-encapsulation process. First, the modeling is validated with hemolysis rates and the drug-loaded quantities to be found in the literature. Then, the variation of the erythrocyte volume, formation of the pore on the erythrocyte membrane, and quantities of drug loaded into and hemoglobin released from erythrocytes are studied. Finally, an optimized operating condition for encapsulating drugs is proposed. The results show that the volume of erythrocytes exposed to hypotonic NaCl solution increases first and then abruptly decreases because of the pore formation; afterwards, it again increases and then decreases slowly. In the presence of the pore, the drug is loaded by diffusion, whereas the leak-induced convection goes against the loading. For an allowed 45% hemolysis rate, with a 10% hematocrit, the optimized NaCl concentration is 0.44%, the optimized time for sealing the loaded erythrocytes with hypertonic NaCl solution is at 6.5 s, and the quantity of albumin (drug) loaded is 4.5 mg/ml cells.

Hazy Weather-Induced Variation in Environmental Behavior of PCDD/Fs and PBDEs in Winter Atmosphere of A North China Megacity.

Haze is a serious pollution problem during the wintertime in North China. In this study, we investigated how the periodic cycle of winter haze events affect the environmental behaviors of two typical persistent organic pollutants, namely, polychlorinated dibenzo- p-dioxins and dibenzofurans (PCDD/Fs) and polybrominated diphenyl ethers (PBDEs), in the atmosphere of a typical megacity, Beijing. The average atmospheric concentrations of the total di- to octa-CDD/Fs (∑PCDD/Fs: 378.0 pg/m3) and the total mono- to nona-BDEs (∑9hPBDEs: 166.5 pg/m3) during haze episodes increased by 3.6-fold and 1.9-fold compared with those during the nonhaze periods, respectively; and their concentrations both linearly increased with PM2.5 levels and decreased as a power function of the atmospheric boundary layer height. The elevated concentrations could be clearly attributed to the vertically sinking motion of airflow in the midlower troposphere. When a haze event occurred, the partitioning rate of PCDD/Fs and PBDEs into particles was reduced; the largest fraction of the particle-bound ∑PCDD/Fs was shifted from ultrafine particles to accumulation mode particles; and a steady-state model (Li-Ma-Yang model) satisfactorily described the gas-particle partitioning of the PCDD/F and PBDE homologues. The inhalation exposure risk evaluation indicated that special attention should be paid to the increased cancer risk induced by the elevated inhalation intake of PCDD/Fs during haze episodes.

Deglycerolization of red blood cells: A new dilution-filtration system.

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.

Unloading of cryoprotectants from cryoprotectant-loaded cells on a microfluidic platform.

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (C G ), the survival rate of cells (S C ) and the recovery rate of cells (R C ) have been studied. Under the designed conditions, C G achieved ~80% and S C reached ~90%. However, R C was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high C G and S C , but low R C . For a low glycerin concentration, C G , S C , and R C were all high. For a high cell density, C G was high, but both S C and R C were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.

A multistage-dialysis microdevice for extraction of cryoprotectants.

In this study, we present a multistage-dialysis microdevice (MDM) for extraction of cryoprotectants (CPAs) from a CPA-laden cell suspension. We confirmed the functions of the key designs of the MDM using a fluorescence solution, we assessed the performance of the MDM by using the MDM to unload glycerin from glycerin-loaded swine erythrocytes, and we investigated the effects of the cell suspension flow rate, glycerin concentration, cell density, and membrane pore size on the clearance efficiency of glycerin (CG), the survival rate of cells (SC), and the recovery rate of cells (RC). Under the designed conditions, CG, SC, and RC reached ~60%, ~90%, and ~70%, respectively. In addition, a high flow rate causes high SC and RC but a low CG. For a low glycerin concentration, CG, SC, and RC are all high. If a low cell density or a large pore membrane is used, CG is high, whereas both SC and RC are low. This work provides insight into the development of microfluidic devices for the inline extraction of cryoprotectants from a small volume of cryopreserved cells prior to the use of the cells in lab-on-a-chip applications.

Staphylococcus epidermidis ΔSortase A strain elicits protective immunity against Staphylococcus aureus infection.

Staphylococcus aureus and Staphylococcus epidermidis are two of the most significant opportunistic human pathogens, causing medical implant and nosocomial infections worldwide. These bacteria contain surface proteins that play crucial roles in multiple biological processes. It has become apparent that they have evolved a number of unique mechanisms by which they can immobilise proteins on their surface. Notably, a conserved cell membrane-anchored enzyme, sortase A (SrtA), can catalyse the covalent attachment of precursor bacterial cell wall-attached proteins to peptidoglycan. Considering its indispensable role in anchoring substrates to the cell wall and its effects on virulence, SrtA has attracted great attention. In this study, a 549-bp gene was cloned from a pathogenic S. epidermidis strain, YC-1, which shared high identity with srtA from other Staphylococcus spp. A mutant strain, YC-1ΔsrtA, was then constructed by allelic exchange mutagenesis. The direct survival rate assay suggested that YC-1ΔsrtA had a lower survival capacity in healthy mice blood compare with the wild-type strain, indicating that the deletion of srtA affects the virulence and infectious capacity of S. epidermidis YC-1. YC-1ΔsrtA was then administered via intraperitoneal injection and it provided a relative percent survival value of 72.7 % in mice against S. aureus TC-1 challenge. These findings demonstrate the possbility that YC-1ΔsrtA might be used as a live attenuated vaccine to produce cross-protection against S. aureus.

Quantification of Short-Chain Chlorinated Paraffins by Deuterodechlorination Combined with Gas Chromatography-Mass Spectrometry.

Analysis of short-chain chlorinated paraffins (SCCPs) is extremely difficult because of their complex compositions with thousands of isomers and homologues. A novel analytical method, deuterodechlorination combined with high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS), was developed. A protocol is applied in the deuterodechlorination of SCCPs with LiAlD4, and the formed deuterated n-alkanes of different alkane chains can be distinguished readily from each other on the basis of their retention time and fragment mass ([M](+)) by HRGC-HRMS. An internal standard quantification of individual SCCP congeners was achieved, in which branched C10-CPs and branched C12-CPs were used as the extraction and reaction internal standards, respectively. A maximum factor of 1.26 of the target SCCP concentrations were determined by this method, and the relative standard deviations for quantification of total SCCPs were within 10%. This method was applied to determine the congener compositions of SCCPs in commercial chlorinated paraffins and environmental and biota samples after method validation. Low-chlorinated SCCP congeners (Cl1-4) were found to account for 32.4%-62.4% of the total SCCPs. The present method provides an attractive perspective for further studies on the toxicological and environmental characteristics of SCCPs.

Atrial overexpression of angiotensin-converting enzyme 2 improves the canine rapid atrial pacing-induced structural and electrical remodeling. Fan, ACE2 improves atrial substrate remodeling.

The purpose of this study was to investigate whether atrial overexpression of angiotensin-converting enzyme 2 (ACE2) by homogeneous transmural atrial gene transfer can reverse atrial remodeling and its mechanisms in a canine atrial-pacing model. Twenty-eight mongrel dogs were randomly divided into four groups: Sham-operated, AF-control, gene therapy with adenovirus-enhanced green fluorescent protein (Ad-EGFP) and gene therapy with Ad-ACE2 (Ad-ACE2) (n = 7 per subgroup). AF was induced in all dogs except the Sham-operated group by rapid atrial pacing at 450 beats/min for 2 weeks. Ad-EGFP and Ad-ACE2 group then received epicardial gene painting. Three weeks after gene transfer, all animals except the Sham group underwent rapid atrial pacing for another 3 weeks and then invasive electrophysiological, histological and molecular studies. The Ad-ACE2 group showed an increased ACE2 and Angiotensin-(1-7) expression, and decreased Angiotensin II expression in comparison with Ad-EGFP and AF-control group. ACE2 overexpression attenuated rapid atrial pacing-induced increase in activated extracellular signal-regulated kinases and mitogen-activated protein kinases (MAPKs) levels, and decrease in MAPK phosphatase 1(MKP-1) level, resulting in attenuation of atrial fibrosis collagen protein markers and transforming growth factor-ß1. Additionally, ACE2 overexpression also modulated the tachypacing-induced up-regulation of connexin 40, down-regulation of connexin 43 and Kv4.2, and significantly decreased the inducibility and duration of AF. ACE2 overexpression could shift the renin-angiotensin system balance towards the protective axis, attenuate cardiac fibrosis remodeling associated with up-regulation of MKP-1 and reduction of MAPKs activities, modulate tachypacing-induced ion channels and connexin remodeling, and subsequently reduce the inducibility and duration of AF.