Detection of cytotoxins by sandwich-ELISA for discrimination of cobra envenomation and indication of necrotic severity.

Snake envenoming is both a healthcare and socioeconomic problem for developing countries and underserved communities. In Taiwan, clinical management of Naja atra envenomation is a major challenge, since cobra venom-induced symptoms are usually confused with hemorrhagic snakebites and current antivenom treatments do not effectively prevent venom-induced necrosis for which early surgical debridement should be administered. Identification and validation of biomarkers of cobra envenomation is critical for progress in setting a realistic goal for snakebite management in Taiwan. Previously, cytotoxin (CTX) was determined as one of potential biomarker candidates; however, its ability to discriminate cobra envenoming remains to be verified, especially in clinical practice. In this study, we selected a monoclonal single-chain variable fragment (scFv) and a polyclonal antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for CTX detection, which successfully recognized CTX from N. atra venom over that from other snake species. Using this specific assay, the CTX concentration in envenoming mice was shown to remain consistent in about 150 ng/mL during the 2-hour post-injection period. The measured concentration was highly correlated with the size of local necrosis in mouse dorsal skin, which the correlation coefficient is about 0.988. Furthermore, our ELISA method displayed 100 % of specificity and sensitivity in discriminating cobra envenoming among snakebite victims through CTX detection and the level of CTX in victim plasma was ranged from 5.8 to 253.9 ng/mL. Additionally, patients developed tissue necrosis at plasma CTX concentrations higher than 150 ng/mL. Thus, CTX not only serves as a verified biomarker for discrimination of cobra envenoming but also a potential indicator of severity of local necrosis. In this context, detection of CTX may facilitate reliable identification of envenoming species and improve snakebite management in Taiwan.

Immunoprofiling of Equine Plasma against Deinagkistrodon acutus in Taiwan: Key to Understanding Differential Neutralization Potency in Immunized Horses.

Snakebite envenoming is a public health issue linked to high mortality and morbidity rates worldwide. Although antivenom has been the mainstay treatment for envenomed victims receiving medical care, the diverse therapeutic efficacy of the produced antivenom is a major limitation. Deinagkistrodon acutus is a venomous snake that poses significant concern of risks to human life in Taiwan, and successful production of antivenom against D. acutus envenoming remains a considerable challenge. Among groups of horses subjected to immunization schedules, few or none subsequently meet the quality required for further scale-up harvesting. The determinants underlying the variable immune responses of horses to D. acutus venom are currently unknown. In this study, we assessed the immunoprofiles of high-potency and low-potency horse plasma against D. acutus venom and explored the conspicuous differences between these two groups. Based on the results of liquid chromatography with tandem mass spectrometry (LC-MS/MS), acutolysin A was identified as the major component of venom proteins that immunoreacted differentially with the two plasma samples. Our findings indicate underlying differences in antivenoms with variable neutralization efficacies, and may provide valuable insights for improvement of antivenom production in the future.

Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells.

The Taiwanese cobra, Naja atra, is a clinically significant species of snake observed in the wild in Taiwan. Victims bitten by N. atra usually experience severe pain and local tissue necrosis. Although antivenom is available for treatment of cobra envenomation, its neutralization potency against cobra-induced necrosis is weak, with more than 60% of cobra envenoming patients developing tissue necrosis after antivenom administration. The present study found that cytotoxin (CTX) is a key component of N. atra venom responsible for cytotoxicity against myoblast cells. Anti-CTX IgY was generated in hens, and the spleens of these hens were used to construct libraries for the development of single chain variable fragments (scFv). Two anti-CTX scFv, S1 and 2S7, were selected using phage display technology and biopanning. Both polyclonal IgY and monoclonal scFv S1 reacted specifically with CTX in cobra venom. In a cell model assay, the CTX-induced cytolytic effect was inhibited only by monoclonal scFv S1, not by polyclonal IgY. Moreover, the neutralization potency of scFv S1 was about 3.8 mg/mg, approximately three times higher than that of conventional freeze-dried neurotoxic antivenom (FNAV). Collectively, these results suggest that scFv S1 can effectively neutralize CTX-induced cytotoxicity and, when combined with currently available antivenom, can improve the potency of the latter, thereby preventing tissue damage induced by cobra envenoming.

Enhanced Paclitaxel Efficacy to Suppress Triple-Negative Breast Cancer Progression Using Metronomic Chemotherapy with a Controlled Release System of Electrospun Poly-d-l-Lactide-Co-Glycolide (PLGA) Nanofibers.

Triple-negative breast cancer (TNBC) is highly aggressive and responds poorly to conventional chemotherapy. The challenge of TNBC therapy is to maximize the efficacies of conventional chemotherapeutic agents and reduce their toxicities. Metronomic chemotherapy using continuous low-dose chemotherapy has been proposed as a new treatment option, but this approach is limited by the selection of drugs. To improve antitumor therapeutic effects, we developed electrospun paclitaxel-loaded poly-d-l-lactide-co-glycolide (PLGA) nanofibers as a topical implantable delivery device for controlled drug release and site-specific treatment. The subcutaneously implanted paclitaxel-loaded nanofibrous membrane in mice was compatible with the concept of metronomic chemotherapy; it significantly enhanced antitumor activity, inhibited local tumor growth, constrained distant metastasis, and prolonged survival compared with intraperitoneal paclitaxel injection. Furthermore, under paclitaxel-loaded nanofiber treatment, systemic toxicity was low with a persistent increase in lean body weight in mice; in contrast, body weight decreased in other groups. The paclitaxel-loaded nanofibrous membranes provided sustained drug release and site-specific treatment by directly targeting and changing the tumor microenvironment, resulting in low systemic toxicity and a significant improvement in the therapeutic effect and safety compared with conventional chemotherapy. Thus, metronomic chemotherapy with paclitaxel-loaded nanofibrous membranes offers a promising strategy for the treatment of TNBC.

Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib.

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SIGNIFICANCE: P. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models.

Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan.

Optimal duration of compression stocking therapy following endovenous thermal ablation for great saphenous vein insufficiency: A meta-analysis.

BACKGROUND: The need for patients to wear compression stockings after varicose vein surgery and the duration of compressions tocking therapy has been debated. This study isa meta-analysis of randomized controlled trials (RCTs) to determine the optimal duration of compression stocking therapy after endovenous thermal ablation (ETA) of the great saphenous vein. METHODS: The PubMed, Embase, and Cochrane Library databases were searched before January 2019. Individual effect sizes were standardized, and a meta-analysis was conducted to calculate the pooled effect size by using a random effects model. The primary outcome was the severity of pain in the postoperative period. Secondary outcomes were quality of life (QoL), leg volume, bruising scores, consumptionof analgesic agents, recovery time off work, satisfaction, and the incidence rates of postoperative complications including paresthesia and phlebitis. RESULTS: Five RCTsinvolving775 patients were reviewed. The long-duration (1-2 weeks) group significantly reduced postoperative pain at 1 week (mean difference [MD] 1.19; 95% confidence interval [CI]: 0.58-1.80) and recovery time off work (MD: 1.01 day, 95% CI: 0.06-1.96)when compared with the short-duration (24-48 h) group. However, the mean pain scores at 2 (0.1; 95% CI: 0-0.2) and 6 weeks postoperatively (-0.3; 95% CI: -1.09-0.49) did not differ significantly between the two groups. Moreover, the incidence rates of complication, paresthesia, and phlebitis did not differ significantly between the short-duration and long-duration groups. CONCLUSION: The use of compression therapy for a long time (1-2 weeks) is better than short-term (24-48 h) use in terms of postoperative pain at 1 week and recovery off work. Hence, we recommend the prescription of 1-week compression stocking therapy after ETA in routine clinical practice. However, the available evidence is of variable quality, further well-structured RCTs with improved standardization of compression treatment, types of stockings, and target populations are warranted.

Snake venom proteome and immuno-profiling of the hundred-pace viper, Deinagkistrodon acutus, in Taiwan.

Deinagkistrodon acutus, also known as the hundred-pace viper or Chinese moccasin, is a clinically significant venomous snake in Taiwan. To address the lack of knowledge on the venom proteome of D. acutus, the venom composition was studied by a bottom-up proteomic approach combining reverse phase high-performance liquid chromatography, SDS-PAGE, and LC-MS/MS analysis. The immunoreactivity and cross-reactivity of Taiwanese freeze-dried D. acutus antivenom (DA-AV) and hemorrhagic antivenom (FH-AV) were investigated, as well. The proteomic analysis revealed the presence of 29 distinct proteins from D. acutus venom belonging to 8 snake venom protein families. Snake venom metalloproteinase (SVMP, 46.86%), C-type lectin (CLEC, 37.59%), phospholipase A2 (PLA2, 7.33%) and snake venom serine protease (SVSP, 6.62%) were the most abundant proteins. In addition to DA-AV, FH-AV also showed a profile of broad immunorecognition toward the venom of D. acutus. Remarkably, both antivenoms specifically reacted with the HPLC fractions containing SVMPs, and the titer was 5-10 times higher than fractions of other components. This information helps us to deeply understand the pathophysiology of D. acutus envenomation and guide us to development of more effective antivenom for clinical treatment.

Dyspnoea in a patient with hepatitis C.

CLINICAL INTRODUCTION: A 60-year-old man had a long history of chronic hepatitis C. He presented to the emergency department with 2 days of progressive dyspnoea. Clinical manifestations included respiratory distress, cyanosis, digital clubbing, spider naevi on the upper chest and shifting dullness in the abdomen (see online supplementary figure S1). The ECG showed sinus tachycardia and left axis deviation. The chest radiography depicted blunting of right costophrenic angle with small pleural effusions and bilateral prominent pulmonary vascular markings (see online supplementary figure S2). The dyspnoea was exacerbated by upright posture and improved with recumbency. The hypoxaemia could not be corrected with administration of 100% oxygen. Contrast-enhanced transthoracic echocardiography was performed with injection of agitated saline (see figure 1 and online supplementary video). CT scan of the chest revealed some vascular abnormalities (see online supplementary figure S3-S6). QUESTION: Which of the following is the next best step in management?Catheter-based closure of intracardiac shuntLiver transplantationPulmonary angiography and embolisationThoracentesisTransjugular intrahepatic portosystemic shunt.

Altered white adipose tissue protein profile in C57BL/6J mice displaying delipidative, inflammatory, and browning characteristics after bitter melon seed oil treatment.

OBJECTIVE: We have previously shown that bitter melon seed oil (BMSO), which is rich in cis-9, trans-11, trans-13 conjugated linolenic acid, is more potent than soybean oil in attenuating body fat deposition in high-fat diet-induced obese C57BL/6J mice. The aim of this study was to obtain a comprehensive insight into how white adipose tissue (WAT) is affected by BMSO administration and to explore the underlying mechanisms of the anti-adiposity effect of BMSO. METHODS AND RESULTS: A proteomic approach was used to identify proteins differentially expressed in the WAT of mice fed diets with or without BMSO for 11 wks. The WAT was also analyzed histologically for morphological changes. Two-dimensional gel electrophoresis (pH 4-7) revealed 32 spots showing a statistically significant difference (P<0.05) in intensity in BMSO-treated mice and 30 of these were shown to code for 23 proteins (15 increased and 8 decreased expression; >2-fold change). Combined with histological evidence of macrophage infiltration and brown adipocyte recruitment, the proteomic and immunoblotting data showed that the WAT in mice subjected to long-term high dose BMSO administration was characterized by reduced caveolae formation, increased ROS insult, tissue remodeling/repair, mitochondria uncoupling, and stabilization of the actin cytoskeleton, this last change being putatively related to an increased inflammatory response. CONCLUSION: The anti-adiposity effect of BMSO is associated with WAT delipidation, inflammation, and browning. Some novel proteins participating in these processes were identified. In addition, the BMSO-mediated WAT browning may account for the increased inflammation without causing adverse metabolic effects.

Outcome of inadequate empirical antibiotic therapy in emergency department patients with community-onset bloodstream infections.

BACKGROUND: Studies on the effect of inadequate empirical antibiotic therapy on the outcome of patients with systemic infection have led to inconsistent results. METHODS: We analysed data from a comprehensive clinical database collected prospectively in a university hospital between 2008 and 2009. All adult patients who registered in the emergency department (ED) with a bloodstream infection (BSI) were enrolled. Empirical therapy was considered adequate if it included antimicrobials to which the specific isolate displayed in vitro susceptibility and that were administered within 24 h of ED admission. The propensity score (PS) was created by a logistic regression model predicting inadequate empirical therapy. PS-adjusted multivariate analysis was performed by the Cox regression model. The Mortality in Emergency Department Sepsis (MEDS) score was used for the adjustment of residual confounding due to differences in the baseline clinical severity of disease. RESULTS: Out of 937 episodes of bacteraemia, 255 (27.2%) patients received inadequate empirical antimicrobial therapy. A crude analysis showed that inadequate antibiotic therapy was associated with higher mortality rates (hazard ratio 1.78, 95% CI 1.30-2.45). PS-adjusted multivariate analyses also showed a significant adverse impact (hazard ratio 1.59, 95% CI 1.14-2.28). The clinical disease severity significantly modified the effect of inadequate antibiotic therapy on survival. The magnitude of the adverse impact of inadequate antibiotic therapy decreased with the increasing severity of sepsis (P=0.009). CONCLUSIONS: Inadequate empirical antimicrobial therapy for community-onset BSI was associated with higher 30 day mortality rates. Study populations with different clinical severities may have different results, which may help to partly explain the heterogeneous findings in many similar studies.

Bitter melon seed oil-attenuated body fat accumulation in diet-induced obese mice is associated with cAMP-dependent protein kinase activation and cell death in white adipose tissue.

The aim of this study was to investigate the antiadiposity effect of bitter melon seed oil (BMSO), which is rich in the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid. In Expt. 1, C57BL/6J mice were fed a butter-based, high-fat diet [HB; 29% butter + 1% soybean oil (SBO)] for 10 wk to induce obesity. They then continued to receive that diet or were switched to an SBO-based, high-fat diet alone (HS; 30% SBO) or containing bitter melon seed oil (BMSO) (HBM; 15% SBO + 15% BMSO) for 5 wk. The body fat percentage was significantly lower in mice fed the HBM diet (21%), but not the HS diet, compared with mice fed the HB diet. In Expt. 2, mice were fed an SBO-based, high-fat diet containing 0 (HS), 5 (LBM), 10 (MBM), or 15% (HBM) BMSO for 10 wk. In the LBM, MBM, and HBM groups, the body fat percentage was significantly lower by 32, 35, and 65%, respectively, compared with the HS control. The reduction in the HBM group was significantly greater than that in the LBM or MBM group. BMSO administration increased phosphorylation of acetyl-CoA carboxylase, cAMP-activated protein kinase (PKA), and signal transducer and activator of transcription 3 in the white adipose tissue (WAT), suggesting that PKA and leptin signaling might be involved in the BMSO-mediated reduction in lipogenesis and increase in thermogenesis and lipolysis. However, compared with the HS control, the HBM group had a significantly higher TNFα concentration in the WAT accompanied by TUNEL-positive nuclei. We conclude that BMSO is effective in attenuating body fat accumulation through mechanisms associated with PKA activation and programmed cell death in the WAT, but safety concerns need to be carefully addressed.

Dietary oxidised frying oil causes oxidative damage of pancreatic islets and impairment of insulin secretion, effects associated with vitamin E deficiency.

We previously reported that, in rodents, a diet with a high oxidised frying oil (OFO) content leads to glucose intolerance associated with a reduction in insulin secretion. The present study aimed at investigating the impairment of pancreatic islets caused by dietary OFO. C57BL/6J mice were divided into three groups to receive a low-fat basal diet containing 5 g/100 g of fresh soyabean oil (LF group) or a high-fat diet containing 20 g/100 g of either fresh soyabean oil (HF group) or OFO (HO group). After 8 weeks, mice in the HO group showed glucose intolerance and hypoinsulinaemia, and their islets showed impaired glucose-stimulated insulin secretion (P < 0·05; HO group v. LF and HF groups). Significantly higher oxidative stress and a lower mitochondrial membrane potential were observed in the islets in the HO group compared with the LF and HF groups. Immunoblots showed that the reduction in insulin levels in HO islets was associated with activation of the c-Jun NH2-terminal kinase and a reduction in levels of pancreatic and duodenal homeobox factor-1. In a second study, when dietary OFO-induced tissue vitamin E depletion was prevented by large-dose vitamin E supplementation (500 IU(1·06 mmol all-rac-α-tocopherol acetate)/kg diet; HO+E group), the OFO-mediated reduction in islet size and impairment of glucose tolerance and insulin secretion were significantly attenuated (P < 0·05; HO group v. HO+E group). We conclude that a high level of dietary OFO ingestion impairs glucose metabolism by causing oxidative damage and compromising insulin secretion in pancreatic islets, and that these effects can be prevented by vitamin E supplementation.