Short-term anaerobic treatment maintained the quality of Actinidia arguta by activating the antioxidant defense system.

BACKGROUND: This study aimed to investigate the effect of 6, 12, and 24 h short-term anaerobic treatment on kiwiberry quality and antioxidant properties at 5 °C. RESULTS: Short-term anaerobic treatment was found to delay ripening and softening in kiwiberries, evident from changes in ethylene release, total soluble solids, starch, protopectin, and fruit texture. The 24 h treatment group exhibited the lowest decay rate of 12% on day 49, a 38% reduction compared with the control group. Anaerobic treatment reduced flesh translucency and decay in the fruit. The 12 h and 24 h treatments enhanced the activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase, and increased the level of total phenolics, flavonoids, anthocyanins, and ascorbic acid. Moreover, it lowered oxidative damage in cell membranes, evidenced by reduced malondialdehyde content and relative conductivity. CONCLUSION: These results indicate that anaerobic treatment maintains the fruit quality by stimulating its antioxidant defense system. Therefore, short-term anaerobic treatment emerges as a promising method for kiwiberry storage. © 2024 Society of Chemical Industry.

Chinese expert consensus on imaging diagnosis of drug-resistant pulmonary tuberculosis.

Tuberculosis (TB) remains one of the major infectious diseases in the world with a high incidence rate. Drug-resistant tuberculosis (DR-TB) is a key and difficult challenge in the prevention and treatment of TB. Early, rapid, and accurate diagnosis of DR-TB is essential for selecting appropriate and personalized treatment and is an important means of reducing disease transmission and mortality. In recent years, imaging diagnosis of DR-TB has developed rapidly, but there is a lack of consistent understanding. To this end, the Infectious Disease Imaging Group, Infectious Disease Branch, Chinese Research Hospital Association; Infectious Diseases Group of Chinese Medical Association of Radiology; Digital Health Committee of China Association for the Promotion of Science and Technology Industrialization, and other organizations, formed a group of TB experts across China. The conglomerate then considered the Chinese and international diagnosis and treatment status of DR-TB, China's clinical practice, and evidence-based medicine on the methodological requirements of guidelines and standards. After repeated discussion, the expert consensus of imaging diagnosis of DR-PB was proposed. This consensus includes clinical diagnosis and classification of DR-TB, selection of etiology and imaging examination [mainly X-ray and computed tomography (CT)], imaging manifestations, diagnosis, and differential diagnosis. This expert consensus is expected to improve the understanding of the imaging changes of DR-TB, as a starting point for timely detection of suspected DR-TB patients, and can effectively improve the efficiency of clinical diagnosis and achieve the purpose of early diagnosis and treatment of DR-TB.

SH3RF2 contributes to cisplatin resistance in ovarian cancer cells by promoting RBPMS degradation.

Platinum-based chemotherapy remains one of the major choices for treatment of ovarian cancer (OC). However, primary or acquired drug resistance severely impairs their efficiency, thereby causing chemotherapy failure and poor prognosis. SH3 domain containing ring finger 2 (SH3RF2) has been linked to the development of cancer. Here we find higher levels of SH3RF2 in the tumor tissues from cisplatin-resistant OC patients when compared to those from cisplatin-sensitive patients. Similarly, cisplatin-resistant OC cells also express higher levels of SH3RF2 than normal OC cells. Through in vitro and in vivo loss-of-function experiments, SH3RF2 is identified as a driver of cisplatin resistance, as evidenced by increases in cisplatin-induced cell apoptosis and DNA damage and decreases in cell proliferation induced by SH3RF2 depletion. Mechanistically, SH3RF2 can directly bind to the RNA-binding protein mRNA processing factor (RBPMS). RBPMS has been reported as an inhibitor of cisplatin resistance in OC. As a E3 ligase, SH3RF2 promotes the K48-linked ubiquitination of RBPMS to increase its proteasomal degradation and activator protein 1 (AP-1) transactivation. Impairments in RBPMS function reverse the inhibitory effect of SH3RF2 depletion on cisplatin resistance. Collectively, the SH3RF2-RBPMS-AP-1 axis is an important regulator in cisplatin resistance and inhibition of SH3RF2 may be a potential target in preventing cisplatin resistance.

A novel automated multi-cycle magnetic solid-phase extraction coupled to LC-MS/MS to study the disorders of six functional B vitamins in patients with gastroenterology and hyperhomocysteinemia.

B vitamins are essential for human life and their disorders can cause a variety of diseases. Solid-phase extraction (SPE) coupled to LC-MS/MS is a preferred technique for determining multiple B vitamins, however, their complexity in real biological matrices makes it hard to achieve satisfactory recovery and accuracy when simultaneous detection. In this study, a novel automated multi-cycle magnetic SPE (MSPE) coupled to the LC-MS/MS method was established using a mixed-mode anion exchange magnetic adsorbent for the simultaneous extraction of six functional B vitamins, including methylmalonic acid, riboflavin, pantothenic acid, 4-pyridoxic acid, folic acid, and 5-methyltetrahydrofolate. After three consecutive MSPE cycles, the recoveries of all analytes were between 51.5% and 89.6%. The method exhibited excellent sensitivity and linearity, with a dynamic range of 200-fold (R > 0.99 for all analytes), exceptional accuracy (ranging between 95.4% and 105.6%) and precision (with RSDs ≤ 6.2%) without significant matrix effects or interferences. Compared to manual SPE method, the automated multi-cycle MSPE method has better feasibility and greater vitamin coverage. It shows a high correlation with the manual method for the detection of 5-methyltetrahydrofolate and folate (R > 0.99). A study of patients from the gastroenterology department showed that those undergoing surgery and those with malignancies may be at risk of folate deficiency. In addition, patients with hyperhomocystinemia had higher levels of methylmalonic acid and lower levels of 5-methyltetrahydrofolate, which correlated with homocysteine levels (R = 0.404 and -0.311, respectively) and showed dose-response relationships. This method is highly automated and cost-effective, with minimal systematic error, making it suitable for the analysis of clinical samples.

Evaluation of pathogen distribution and antimicrobial resistance in breast plastic surgery infection.

BACKGROUND: The demand for mammaplasty has increased in recent years, and infection remains one of the common and serious post-operative complications. In this study, we analyzed the pathogen distribution and antibiotic susceptibility of breast plastic surgery infections, and compared the differences in pathogenic species between surgical procedures. METHODS: The number of each species was counted in the microbial samples of breast plastic surgery infections in Plastic Surgery Hospital of Chinese Academy of Medical Sciences from January 2011 to December 2021. The in vitro antibiotic sensitivity testing data were analyzed using WHONET 5.6 software. The surgical techniques, the period of infection, and other details were gathered in accordance with the clinical data. RESULTS: There were a total of 42 cases included, and 43 different types of pathogenic bacteria, mostly gram-positive bacteria, were found. CoNS (13/43) and Staphylococcus aureus (22/43) made up the majority. The most prevalent of the five Gram-negative bacteria was Pseudomonas aeruginosa. Results of drug sensitivity tests indicate that S. aureus is highly sensitive to vancomycin, cotrimoxazole, and linezolid, whereas CoNS is highly sensitive to vancomycin, linezolid, and chloramphenicol. Both of these bacteria show high resistance to erythromycin and penicillin. Breast augmentation, breast reconstruction, and breast reduction surgery were the most frequently associated breast surgery procedures in this study with infections, with the highest number of infections occurring following breast augmentation with fat grafting, breast reduction surgery, and breast reconstruction with autologous tissue. Various breast plastic surgery procedures have different common pathogens of infection, but the most prevalent are CoNS and S. aureus. Additionally, the majority of the infections in this study were in the early stages. CONCLUSIONS: Gram-positive bacteria were the predominant cause of breast plastic surgery infections, and the types of infection strains, the period of infection onset, and the antibiotic susceptibility of prevalent strains varied between breast plastic procedures.

Efficacy and safety of tocilizumab and baricitinib among patients hospitalized for COVID-19: a systematic review and meta-analysis.

Introduction: Tocilizumab and baricitinib are recommended treatment options for COVID-19 patients with hyperinflammatory response; however, there is a lack of systematic review directly evaluating their efficacy and safety. Objective: This review was conducted to evaluate the efficacy and safety of tocilizumab and baricitinib in the treatment of hospitalized patients with COVID-19. Methods: Relevant databases were searched for studies that compared the effect or safety of baricitinib or tocilizumab in hospitalized patients with COVID-19. The mortality was the main outcome. The hospital length of stay or adverse drug reactions were taken into consideration as secondary endpoints. The analyses were performed in Revman 5.3 or Stata 16.0. The protocol and analysis plan were pre-registered in PROSPERO, with the registration number CRD42023408219. Results: In total, 10 studies with 2,517 patients were included. The overall pooled data demonstrated that, there was no statistically significant difference in the 28-day mortality rate and the hospital length of stay between the tocilizumab and baricitinib (OR = 1.10, 95% CI = 0.80-1.51, p = 0.57; OR = -0.68, 95% CI = -2.24-0.87, p = 0.39). The adverse reactions including secondary infection rate, thrombotic and bleeding events, and acute liver injury of tocilizumab were significantly higher than that of baricitinib. (OR = 1.49, 95% CI = 1.18-1.88, p < 0.001,OR = 1.52, 95% CI = 1.11-2.08, p = 0.009; OR = 1.52, 95% CI = 1.11-2.08, p = 0.009; OR = 2.24, 95% CI = 1.49-3.35, p < 0.001). Conclusion: In patients hospitalized with COVID-19, no discernible difference in therapeutic efficacy was observed between tocilizumab and baricitinib; however, the group treated with baricitinib demonstrated a significantly lower incidence of adverse effects.

Distribution of Pathogenic Bacteria and Antimicrobial Resistance after Plastic Surgery for Microtia.

Background: Microtia, or congenital malformation (smallness or absence) of the outer ear, can be treated with ear prosthetics and/or surgery. Methods: Between January 2011 and December 2021, following plastic surgery, microbial strains from patients with microtia were collected, identified, and counted. WHONET 5.6 was used to analyze in vitro drug resistance of the microbial strains, according to procedures outlined by the Clinical and Laboratory Standards Institute (document M100, 2021). Data regarding surgical techniques, the duration of infection, and other clinical details were also collected. Results: A total of 261 patients were included in the study. Among these, 235 Gram-positive bacteria were detected, with Staphylococcus aureus (140/235) and coagulase-negative staphylococci (84/235) accounting for the majority. There were also 26 Gram-negative bacteria, of which Enterobacter (11/26) and Pseudomonas aeruginosa (7/26) were the most common. According to the results of testing for antimicrobial resistance, S. aureus was highly sensitive to cotrimoxazole, levofloxacin, vancomycin, chloramphenicol, and linezolid, whereas coagulase-negative staphylococci were highly sensitive to vancomycin and linezolid. Both were highly resistant to penicillin and erythromycin. In this study, the pathogenic bacteria involved in postoperative infections varied overall, but the most prevalent was S. aureus. The infections appeared mainly in the late postoperative period. A total of 24,548 procedures were performed in the same period, and the infection rate was 1.06%. Conclusions: Gram-positive bacteria are the major cause of infection following plastic surgery for microtia. The bacterial species, degrees of antimicrobial resistance, and length of infection varied among the various surgical procedures.

Higher incidence of hematuria was observed in female children with microtia.

The goals of this study were to investigate the incidence and characteristics of hematuria in patients with microtia, and to clarify that more attention should be paid to renal dysfunction in patients with microtia. We conducted a retrospective cohort study of a total 9447 children diagnosed with microtia (selected as study group, 7037 children) or pigmented nevus (selected as control group, 2410 children) at the Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from January 2009 to June 2021. All of the routine urinalysis report of these children were reviewed to assess the incidence and characteristics of hematuria in each group. No statistically significant differences were observed when analyzing the overall incidence of hematuria between the study and control groups (P > 0.05). However, after grouping by sex, the incidence of hematuria in female children with microtia was significantly higher than that in femalecontrol group and no similar results were observed in the male patients. In addition, after further grouping by age in case group, the incidence of hematuria in girls of all ages with microtia was significantly higher than that in males with microtia (age 0-10:males: Girls = 1.89%:4.14%; age 0-5: males: Girls = 1.22%:3.73%; age 6-10: males:Girls = 1.97%:4.14%,P < 0.05), while no similar results were obtained in the control group.(age 0-10:males: Girls = 1.39%:2.22%; age 0-5: males: Girls = 1.07%:1.95%; age 6-10: males: Girls = 3.38%:3.17%, P > 0.05). Higher incidence of hematuria was observed in female children with microtia.

Analysis of pathogen distribution and antimicrobial resistance at infected sites in plastic surgery.

OBJECTIVE: By analyzing the distribution and drug resistance of common pathogen in different sites in plastic surgery to provide reference for clinicians to choose the best antibacterial treatment plan. METHODS: Pathogens of postoperative infection in plastic surgery from January 2011 to December 2021 were retrospectively analyzed to determine the species and quantity, and to access the trend of each pathogen's detection rate. The antibiotic sensitivity and distribution characteristics of common pathogens were studied in conjunction with the site of infection. RESULTS: A total of 1709 bacterial strains were detected, including 1244 gram-positive bacterial strains and 465 gram-negative bacterial strains. The main pathogen of perineum was Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while Staphylococcus aureus (S. aureus) was the most common pathogen in the other infected sites. The detection rate of methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococcus (MRCNS) was on the rise from 2011 to 2021. No S. aureus and coagulase-negative staphylococcus (CoNS) strains were resistant to vancomycin. The sensitive rate of S. aureus from all parts and CoNS from all sites except lower limbs and mandible was higher than 80% to linezolid. The resistance rate of S. aureus and CoNS in all parts to penicillin, clindamycin, and erythromycin was high. The susceptibility rate of CoNS in lower mandible was high to gentamicin. CONCLUSIONS: Staphylococcus aureus was the primary pathogen of gram-positive bacteria in all site of plastic surgery except perineum, followed by CoNS. The distribution and drug resistance of pathogen in different infection sites were different. We should formulate more accurate and reasonable antibacterial programs according to drug resistance results of various parts to reduce the emergence of resistant strains and effectively prevent and control infection.

Simple and fast determination of tetrodotoxin in human plasma based on hydrophilic-interaction/ion-exchange mixed-mode solid phase extraction combined with liquid chromatography-tandem mass spectroscopy.

In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the measurement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant matrix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic interaction and ion exchange. 100µL plasma sample was precipitated rapidly with acetonitrile containing 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed with 95% acetonitrile, TTX was eluted with 200µL of 50% acetonitrile containing 1% trichloroacetic acid. 2µL of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide column was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and timesaving (<30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using m/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transition for TTX) and 179.2→63.0 (transition for IS), respectively. The method was linear in the range of 0.1-20 ng/mL for TTX with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra- and inter-assay accuracies were in the range of 98.5%-99.8% (relative standard deviations, RSDs ≤ 5.92%) and 98.8-99.5% (RSDs ≤ 6.23%), respectively. Biases of spiking analysis were ranged from -7.00% to 7.43% for healthy human plasma samples (RSDs ≤ 8.83%) and from -5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high bilirubin plasma samples (RSDs ≤ 6.40%), which proved the good anti-interference property of the method. The results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.

Development and Validation of a Nomogram to Predict the Risk of Blood Transfusion in Orthognathic Patients.

OBJECTIVE: This study aims to establish a nomogram to predict the probability of blood transfusion in patients with preoperative autologous blood donation before orthognathic surgery. METHODS: The authors conducted a retrospective case-control study on consecutive orthognathic patients with preoperative autologous blood donation from January 2014 to December 2020. The outcome variable was the actual transfusion of autologous blood (ATAB). Predictors included patients' demographics, preoperative blood cell test, vital signs, American Society of Anesthesiologists classification, surgical procedure, operation duration, and blood loss. Univariable and multivariable logistic regressions were performed to identify independent risk factors associated with ATAB. A nomogram was constructed to predict the risk for ATAB. The performance of the nomogram was evaluated using the area under the receiver operating characteristic curve, calibration curve and the consistency index. RESULTS: A total of 142 patients (75 males and 67 females) with an average age of 22.72 ± 5.34 years donated autologous blood before their orthognathic surgery. Patients in the transfusion group (n = 56) had significantly lower preoperative red blood cell counts (4.74 ± 0.55 × 10 9 /L versus 4.98 ± 0.45 × 10 9 /L, P = 0.0063), hemoglobin (141.48 ± 15.18g/dL versus 150.33 ± 14.73g/dL, P = 0.0008), and hematocrit (41.05% ± 4.03% versus 43.32% ± 3.42%, P = 0.0006), more bimaxillary osteotomies (92.86% versus 56.98%, P < 0.001), longer operation duration (348.4 ± 111.10 minutesversus261.6 ± 115.44 minutes, P < 0.001), and more intraoperative blood loss (629.23±273.06 ml versus 359.53 ± 222.84 ml, P < 0.001) than their counterparts (n = 86) in the non- transfusion group. Univariable and multivariable logistic regression demonstrated that only hemoglobin (adjusted odds ratio [OR] 0.864, 95% confidence interval [CI]:0.76-0.98, P = 0.026), operation procedures (adjusted OR 8.14, 95% CI:1.69-39.16, P = 0.009), and blood loss (adjusted OR 1.006, 95% CI:1.002-1.009, P < 0.001) were independent risk factors for ATAB. The area under the receiver operating characteristic curve of the nomogram was 0.823. The consistency index of the nomogram was 0.823. The calibration curve illustrated that the nomogram was highly consistent with the actual observation. CONCLUSIONS: The nomogram is a simple and useful tool with good accuracy and performance in predicting the risk for blood transfusion.

Granzyme B and perforin produced by SEC2 mutant-activated human CD4+ T cells and CD8+ T cells induce apoptosis of K562 leukemic cells by the mitochondrial apoptotic pathway.

Staphylococcal enterotoxin C2 (SEC2), a classical representative of superantigens, activates T cells that produce massive cytokines. This characteristic makes SEC2 a promising candidate drug for cancer immunotherapy. Previous study showed that ST-4, a SEC2 mutant, enhanced recognition of mouse T-cell receptor Vß regions, and activated the increased number of T cells that produced more cytokines. However, the underlying molecular mechanism for stimulation of human peripheral blood mononuclear cells (PBMCs) and antitumor effect on human tumor cells remains unknown. Herein, we showed that ST-4 significantly activated TCR Vß 12, 13A, 14, 15, 17, and 20 CD4+ and CD8+ T cells, which produced substantial amounts of granzyme B and perforin. These cytokines exhibited antitumor effect on K562 cells by promoting apoptosis and inducing S-phase cell cycle arrest. Conversely, the granzyme B inhibitor or perforin inhibitor significantly weakened antitumor effect of ST-4, accompanied by a decrease of cleaved proapoptotic BAX and cytochrome c, and an increase of antiapoptotic BCL2. Taken together, these data suggest that granzyme B and perforin produced by ST-4-activated CD4+ T cells and CD8+ T cells play a pivotal role in inducing K562 cell apoptosis by the mitochondrial apoptotic pathway, and support ST-4 as a potential candidate for cancer immunotherapy.

Clinical characteristics and changes of chest CT features in 307 patients with common COVID-19 pneumonia infected SARS-CoV-2: A multicenter study in Jiangsu, China.

OBJECTIVE: The study was aimed to describe the clinical characteristics and evaluate the dynamic changes of chest CT features in the first three weeks in the common type fo COVID-19 pneumonia patients in Jiangsu Province. METHODS: 307 patients infected SARS-CoV-2 classified as common type were enrolled in the study. 628 chest CT scans were divided into three groups based on the time interval between symptoms and chest CT scan. The clinical characteristics were descriptively analyzed.The chest CT features were quantitatively evaluated. Mann-Whitney U test was used to test the differences in three groups and between men and women. Spearman rank correlation was used to test the association between the arterial blood gas(ABG) analysis results and chest CT scores. RESULTS: Fever (69.1%) and cough (62.8%) were common symptoms. 111(36.2%) patients were anorexia. GGO was the most common manifestation of COVID-19 pneumonia, which could be followed by consolidation and fibrosis. Lower lobe or subpleural region was the most common distribution form of lesion. More lung lobes were involved in the third week. Total chest CT scores in the second week were higher than the first week. Fibrosis Scores increased in the second and third week. Total CT score, GGO score and fibrosis score of male patients were significantly higher than female in the second week. Male patients had higher consolidation score and fibrosis score than female in the third week. Total CT score and GGO score had weak to moderate correlation with arterial blood gas indices. CONCLUSION: Changes in chest CT were difficult to assess quantitatively in the first third weeks. Male patients recovered slower than female in the second week. Although CT score had correlations with arterial blood gas indices, long-term follow-up of pulmonary function test is needed to determine the recovery of lung.

Antibiotic-impregnated bone cement for preventing infection in patients receiving primary total hip and knee arthroplasty: A meta-analysis.

BACKGROUND: Surgical-site infections after primary total joint arthroplasty (TJA) are a significant issue. Antibiotic-impregnated bone cement (AIBC) has been widely used for the treatment of infected joints, but routine use of AIBC in primary TJA remains controversial. In this systematic review, we evaluated the efficacy of AIBC in reducing surgical-site infections after primary TJA. METHODS: We systematically searched Pubmed, EMbase, Cochrane Library, CMB, CNKI, and WanFang Data for studies (published until June 1, 2019) evaluating AIBC use in reducing infection rates. Two reviewers independently screened the literature according to inclusion and exclusion criteria, extracted data, and assessed the methodological quality of included studies. Meta-analysis was performed using Review Manager 5.3 software. The registration number is CRD42017078341 in PROSPERO. RESULTS: In total, 10 studies were included, resulting in a sample size of 13,909 arthroplasty cases. The overall pooled data demonstrated that, compared with systemic antibiotics, AIBC was more effective in decreasing deep infection rates (odds ratio [OR] = 0.35, 95% confidence interval [CI] = 0.14-0.89, P = .030), although there were higher superficial infection rates with AIBC (OR = 1.53, 95% CI = 1.11-2.11, P = .010). Compared to systemic antibiotics alone, AIBC with systemic antibiotics significantly decreased deep infection rates (OR = 0.55, 95% CI = 0.41-0.75, P = .0001) but there was no difference in superficial infection rates (OR = 1.43, 95% CI = 0.81-2.54, P = .220). In the subgroup analysis, both randomized controlled trials and cohort studies had reduced deep infection rates after primary TJA (OR = 0.61, 95% CI = 0.37-0.99, P = .050 and OR = 0.49, 95% CI = 0.34-0.70, P = .0001, respectively). AIBC decreased deep infection rates in both total hip and knee arthroplasty (OR = 0.25, 95% CI = 0.12-0.52, P = .0002 and OR = 0.62, 95% CI = 0.45-0.87, P = .005, respectively). Deep infection rates were significantly decreased by AIBC with gentamicin (OR = 0.31, 95% CI = 0.20-0.49, P < .00001) but unaffected by AIBC with cefuroxime (OR = 0.35, 95% CI = 0.10-1.20, P = .100). Deep infection rates in the AIBC and control groups were similar when laminar airflow was applied to the operating room (OR = 0.90, 95% CI = 0.60-1.35, P = .620); however, without laminar airflow, the efficacy of AIBC in decreasing deep infection rates was significantly higher than that of control group (OR = 0.21, 95% CI = 0.08-0.59, P = .003). CONCLUSIONS: AIBC may significantly decrease deep infection rates after primary total hip and knee arthroplasty, with or without systemic antibiotics.

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33-/--null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33-/- mice from ß-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

Deposition of diazepam and its metabolites in hair following a single dose of diazepam.

Only sporadic data are available on hair concentrations of diazepam and some of its metabolites (nordazepam, oxazepam, and temazepam) following a single controlled dose. The aim of this study was to investigate the deposition of diazepam and its metabolites in human hair after eight healthy volunteers (four women and four men, ages 24-26, East Asian) consumed 10 mg of diazepam. Hair was collected from all volunteers 1 month after exposure, and also 2 months post-exposure from men and 10 months post-exposure from women. Diazepam and the complete metabolite profile, including oxazepam glucuronide and temazepam glucuronide, were measured by ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with limits of quantifications (LOQs) of 0.5-2.5 pg/mg for diazepam, nordazepam, oxazepam, and temazepam, and of 10 pg/mg for oxazepam glucuronide and temazepam glucuronide. There were no differences by gender in the amounts of diazepam or metabolites found. The concentration of the main metabolite nordazepam was consistently higher than that of diazepam at both 1 and 2 months after consumption. Oxazepam and temazepam traces were found in some volunteers' hair, but the glucuronides were not detected. Diazepam and nordazepam levels at 10 months post-exposure were extremely low (near the LOQ), indicating drug loss by personal hygiene and physical handling. To our knowledge, this is the first single-dose diazepam study using black hair and the first study to include measurements of oxazepam glucuronide and temazepam glucuronide in human hair.

Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid-phase extraction and liquid chromatography with tandem mass spectrometry.

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 µL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

Determination of co-administrated opioids and benzodiazepines in urine using column-switching solid-phase extraction and liquid chromatography-tandem mass spectrometry.

Co-administration of opioids with benzodiazepines is very common around the world. A semi-automated method was developed for the determination of four opioids and two benzodiazepines as well as their metabolites (including glucuronide metabolites) in human urine, based on on-line column-switching-solid-phase extraction (CS-SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CS-SPE was performed by loading 200µL of urine sample to an Oasis HLB cartridge. Detection was achieved using a LC-MS/MS system equipped with an electrospray ionization source (ESI). For unequivocal identification and confirmation, two selected reaction monitoring transitions were registered for each compound, and no co-elution of interferences was observed at the expected retention time. Significant ion suppressions were observed for most analytes during chromatographic runs, but isotope-labeled internal standards (ISs) were used and found to be useful to compensate for the determination error caused by the matrix effect. The assay's linearity ranged from 1-20ng/mL to 800-1000ng/mL for 23 compounds, except for lorazepam (LOR), whose linearity was in the range of 1-100ng/mL. This method showed to be precise and accurate. The relative standard deviation (RSD) % values of within-run precision, between-run precision and total precision were not greater than 10.4% (n=3), 12.9% (n=5) and 15.1% (n=15), respectively. Accuracy values were in the range of 87.5-110%. Limits of detection (LODs) ranged from 0.2ng/mL to 5ng/mL, and limits of quantification (LOQs) ranged from 1ng/mL to 20ng/mL. The method was applied to the assay of 12 samples from forensic cases, which exemplified the co-administration of benzodiazepines (BZDs) by some heroin abusers. This method was of high sensitivity, selectivity and reliability, minimum sample manipulation, semi-automation, and fairly high throughput (analysis time per sample was 20min). The method developed will be useful for the detection of co-administrated drugs and the study of the interactions of BDZs with opioids.