Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

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.

Novel Pyrazole Amides as Potential 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an important target for the development of new herbicides. HPPD inhibitors can hinder photosynthesis and induce weed death with bleaching symptoms. To explore the novel skeleton of HPPD inhibitors, a series of novel pyrazole amide derivatives were synthesized and evaluated for their inhibitory effects on Arabidopsis thaliana HPPD (AtHPPD) and herbicidal activities. Some compounds had excellent inhibitory activities against AtHPPD. Among them, compound B5 displayed top-rank inhibitory activity against AtHPPD with an IC50 value of 0.04 µM, which was obviously superior to that of topramezone (IC50 value of 0.11 µM). Furthermore, compounds B2 and B7 had 100% herbicidal activities in Petri dish assays against Portulaca oleracea and Amaranthus tricolor at 100 µg/mL. In particular, compound B7 not only possessed strong AtHPPD inhibitory activity but also exhibited significant preemergence herbicidal activity. However, compound B7 was completely harmless to soybean, cotton, and wheat. In addition, the molecular docking and microscale thermophoresis measurement experiment verified that compounds can bind well with AtHPPD via π-π interactions. The present work provides a new approach for the rational design of more effective HPPD inhibitors, and pyrazole amides could be used as useful substructures for the development of new HPPD inhibitors and preemergence herbicidal agents.

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.

Abdominal Tuberculosis Managed Surgically in the Late Phase: A Case Report.

About 1-5 % of cases of tuberculosis (TB) have uncommon abdominal conditions, and affect primarily young adults. The clinical diagnosis is challenging and often delayed due to the symptoms being non-specific and may be confused with other bowel diseases, therefore resulting in significant morbidity and mortality. A 27-year-old man was brought to our emergency department with the complaints a fever and abdominal pain. According to the chest X-ray findings taken 24 hours previously, pulmonary TB was suspected. Abdominal TB associated intestinal obstruction, ascites and lymphadenopathy were found by computed tomography and exploratory laparotomy. He was treated surgically by segmental resection, however passed away due to severe bleeding caused by the recurrence of perforation of the small intestine and sepsis. This case of systemic TB highlights the difficulties in diagnosis and treatment in time. The early diagnosis and timely treatment are both necessary to manage the disease successfully.

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.

Increased risk of pyogenic liver abscess in patients with alcohol intoxication: A population-based retrospective cohort study.

We designed a population-based retrospective cohort study to investigate the association between the event of alcohol intoxication and the risk of pyogenic liver abscess. The present study enrolled 245,076 patients with a history of alcohol intoxication from 2000 to 2010 and matched each of them with four comparison patients, with similar mean age and sex ratios. We determined the cumulative incidences and adjusted hazard ratios (aHRs) of liver abscess. A significant association was observed between alcohol intoxication and liver abscess. The incidence density rate of liver abscess was 3.47-fold greater in the alcohol intoxication (AI) cohort than in the non-AI cohort (12.2 vs. 3.43 per 10,000 person-years), with an adjusted HR (aHR) of 2.64 (95% CI = 2.26 to 3.08). This population-based study positively associated the event of alcohol intoxication with increased risk of liver abscess. Our findings warrant further large-scale and in-depth investigations in this area.

Relationship Between Acute Benzodiazepine Poisoning and Acute Pancreatitis Risk: A Population-Based Cohort Study.

We designed a population-based retrospective cohort study to investigate the association between the event of benzodiazepine (BZD) poisoning and the risk of acute pancreatitis.In the present study, 12,893 patients with BZD poisoning during 2000 to 2011 were enrolled and matched with 4 comparison patients according to mean age and sex. We determined the cumulative incidences and adjusted hazard ratios of acute pancreatitis.A significant association was observed between BZD poisoning and acute pancreatitis. After adjustment for potential risk factors, the patients with BZD poisoning had a 5.33-fold increased risk of acute pancreatitis compared with the controls without BZD poisoning (HR = 5.33, 95% CI = 2.26-12.60). The results revealed that acute pancreatitis in patients with BZD poisoning occurred in a follow-up time of ≤1 month (HR = 50.0, P < .001), and the risk of acute pancreatitis was no different between the patients with and without BZD poisoning when the follow-up time was >1 month (HR = 1.07, P > .05).This population-based study revealed the positive correlation between the event of BZD poisoning and an increased risk of acute pancreatitis. The findings warrant further large-scale and in-depth investigation.

Fabrication and biosensing with CNT/aligned mesostructured silica core-shell nanowires.

We report the synthesis of carbon nanotubes (CNTs)/mesostructured silica core-shell nanowires via an interfacial surfactant templating approach. The nanowires possess perpendicularly aligned and uniform accessible mesopores, high surface area and large pore volume. When dimethyl sulfoxide reductase (DMSOR) enzyme is immobilized on the core-shell nanowires, the complex can enhance the electrical communication between the active sites of the enzyme and the electrode surface in the presence of a mediator. The unique properties of the CNTs and the uniform accessible mesopores of the nanowires have made this material promising in the applications as carbon nanotubes field-effect transistors, electrochemical detection, and biosensors.