Development and optimization of a DNA aptamer to delay ß-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. ß-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against ß-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for ß-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'-O-methylation and a 3'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for ß-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of ß-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

Development of antibody-detection ELISA based on beta-bungarotoxin for evaluation of the neutralization potency of equine plasma against Bungarus multicinctus in Taiwan.

Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, ß-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, ß-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on ß-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A2 (PLA2) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production.

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.

Taiwan cobra envenoming: serum venom concentration before and after specific treatment and relationship with debridement of necrotic wound tissue.

Background: Bivalent freeze-dried neurotoxic (FN) antivenom has been the primary treatment since the 1980s for Taiwan cobra (Naja atra) envenomation in Taiwan. However, envenomation-related wound necrosis is a significant problem after cobra snakebites. In the present study, we analyzed the changes in serum venom concentration before and after antivenom administration to discover their clinical implications and the surgical treatment options for wound necrosis. Methods: The patients were divided into limb swelling and wound necrosis groups. The clinical outcome was that swelling started to subside 12 hours after antivenom treatment in the first group. Serum venom concentrations before and after using antivenoms were measured to assess the antivenom's ability to neutralize the circulating cobra venom. The venom levels in wound wet dressing gauzes, blister fluids, and debrided tissues were also investigated to determine their clinical significance. We also observed the evolutional changes of wound necrosis and chose a better wound debridement timing. Results: We prospectively enrolled 15 Taiwan cobra snakebite patients. Males accounted for most of this study population (n = 11, 73%). The wound necrosis group received more antivenom doses than the limb swelling group (4; IQR:2-6 vs 1; IQR:1-2, p = 0.05), and less records of serum venom concentrations changed before/after antivenom use (p = 0.0079). The necrotic wound site may release venom into circulation and cause more severe envenomation symptoms. Antivenom can efficiently diminish limb swelling in cobra bite patients. However, antivenom cannot reduce wound necrosis. Patients with early debridement of wound necrosis had a better limb outcome, while late or without debridement may have long-term hospital stay and distal limb morbidity. Conclusions: Antivenom can efficiently eliminate the circulating cobra venom in limb swelling patients without wound necrosis. Early debridement of the bite site wound and wet dressing management are suggestions for preventing extended tissue necrosis and hospital stay.

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.

The Clinical Usefulness of Taiwan Bivalent Freeze-Dried Hemorrhagic Antivenom in Protobothrops mucrosquamatus- and Viridovipera stejnegeri-Envenomed Patients.

Snakebites from Protobothrops mucrosquamatus (Taiwan habus) and Viridovipera stejnegeri (green bamboo vipers) account for the most venomous snakebites in Taiwan. The bivalent freeze-dried hemorrhagic (FH) antivenom is employed to treat these two snakebite patients without a strict clinical trial. We evaluated the clinical usefulness of Taiwan bivalent freeze-dried hemorrhagic (FH) antivenom in Taiwan habu- and green bamboo viper-envenomed patients. We checked ELISA- based serum venom antigen levels before and after FH antivenom to evaluate FH's ability to neutralize patients' serum snake venom and its usefulness in reducing limb swelling after snakebites. Patients who had higher serum venom antigen levels had more severe limb swelling. Of the 33 enrolled patients, most of their snake venom antigen levels were undetected after the appliance of antivenom. Most enrolled patients (25/33) had their limb swelling subside within 12 h after antivenom treatment. The failure to reduce limb swelling was probably due to an inadequate antivenom dose applied in more severely envenomated patients. Our data indicate the feasibility of the FH antivenom in effectively eliminating venom and resolving the affected limb swelling caused by Taiwan habu and green bamboo viper bites.

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.

Development of Antibody Detection ELISA Based on Immunoreactive Toxins and Toxin-Derived Peptides to Evaluate the Neutralization Potency of Equine Plasma against Naja atra in Taiwan.

Naja atra, also known as Taiwanese cobra, is one of the most prevalent venomous snakes in Taiwan. Clinically, freeze-dried neurotoxic antivenom (FNAV) produced from horses by Taiwan Centers for Disease Control (CDC) has been the only approved treatment for N. atra envenoming for the last few decades. During antivenom production, large numbers of mice are used in the in vivo assay to determine whether the neutralization potency of hyperimmunized equines is satisfactory for large-scale harvesting. However, this in vivo assay is extremely laborious, expensive, and significantly impairs animal welfare. In the present study, we aimed to develop an in vitro ELISA-based system that could serve as an alternative assay to evaluate the neutralization potency of plasma from hyperimmunized equines. We initially obtained 51 plasma samples with known (high or low) neutralization potency assessed in vivo from 9 hyperimmunized equines and subsequently determined their antibody titers against the five major protein components of N. atra venom (neurotoxin (NTX), phospholipase A2 (PLA2), cytotoxin (CTX), cysteine-rich secretory protein (CRISP), and snake venom metalloproteinase (SVMP)) via ELISA. The antibody titer against NTX was the most effective in discriminating between high and low potency plasma samples. To identify the specific epitope(s) of NTX recognized by neutralization potency-related antibodies, 17 consecutive NTX-derived pentadecapeptides were synthesized and used as antigens to probe the 51 equine plasma samples. Among the 17 peptides, immunoreactive signals for three consecutive peptides (NTX1-8, NTX1-9, and NTX1-10) were significantly higher in the high potency relative to low potency equine plasma groups (p < 0.0001). Our ELISA system based on NTX1-10 peptide (RWRDHRGYRTERGCG) encompassing residues 28-42 of NTX displayed optimal sensitivity (96.88%) and specificity (89.47%) for differentiating between high- and low-potency plasma samples (area under the receiver operating characteristic curve (AUC) = 0.95). The collective data clearly indicate that the antibody titer against NTX protein or derived peptides can be used to efficiently discriminate between high and low neutralization potency of plasma samples from venom-immunized horses. This newly developed antibody detection ELISA based on NTX or its peptide derivatives has good potential to complement or replace the in vivo rodent assay for determining whether the neutralization potency of equine plasma is satisfactory for large-scale harvesting in the antivenom production process against N. atra.

Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis.

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims' plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange-high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10-15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

Effects of Sodium Silicate Complex against Hemorrhagic Activities Induced by Protobothrops mucrosquamatus Venom.

Protobothrops mucrosquamatus poses a serious medical threat to humans in Southern and Southeastern Asia. Hemorrhage is one of the conspicuous toxicities related to the pathology of P. mucrosquamatus envenoming. Previous in vitro and in vivo studies showed that a silica-derived reagent, sodium silicate complex (SSC), was able to neutralize hemorrhagic and proteolytic activities induced by pit viper venoms, including Crotalus atrox, Agkistrodoncontortrix contortrix and Agkistrodon piscivorus leucostoma. In this study, we validated that SSC could neutralize enzymatic and toxic effects caused by the venom of P. mucrosquamatus. We found that SSC inhibited the hemolytic and proteolytic activities induced by P. mucrosquamatus venom in vitro. In addition, we demonstrated that SSC could block intradermal hemorrhage caused by P. mucrosquamatus venom in a mouse model. Finally, SSC could neutralize lethal effects of P. mucrosquamatus venom in the mice. Therefore, SSC is a candidate for further development as a potential onsite first-aid treatment for P. mucrosquamatus envenoming.

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.

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.

Development of sandwich ELISA and lateral flow strip assays for diagnosing clinically significant snakebite in Taiwan.

Taiwan is an island located in the south Pacific, a subtropical region that is home to 61 species of snakes. Of these snakes, four species-Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Bungarus multicinctus and Naja atra-account for more than 90% of clinical envenomation cases. Currently, there are two types of bivalent antivenom: hemorrhagic antivenom against the venom of T. stejnegeri and P. mucrosquamatus, and neurotoxic antivenom for treatment of envenomation by B. multicinctus and N. atra. However, no suitable detection kits are available to precisely guide physicians in the use of antivenoms. Here, we sought to develop diagnostic assays for improving the clinical management of snakebite in Taiwan. A two-step affinity purification procedure was used to generate neurotoxic species-specific antibodies (NSS-Abs) and hemorrhagic species-specific antibodies (HSS-Abs) from antivenoms. These two SSAbs were then used to develop a sandwich ELISA (enzyme-linked immunosorbent assay) and a lateral flow assay comprising two test lines. The resulting ELISAs and lateral flow strip assays could successfully discriminate between neurotoxic and hemorrhagic venoms. The limits of quantification (LOQ) of the ELISA for neurotoxic venoms and hemorrhagic venoms were determined to be 0.39 and 0.78 ng/ml, respectively, and the lateral flow strips were capable of detecting neurotoxic and hemorrhagic venoms at concentrations lower than 5 and 50 ng/ml, respectively, in 10-15 min. Tests of lateral flow strips in 21 clinical snakebite cases showed 100% specificity and 100% sensitivity for neurotoxic envenomation, whereas the sensitivity for detecting hemorrhagic envenomation samples was 36.4%. We herein presented a feasible strategy for developing a sensitive sandwich ELISA and lateral flow strip assay for detecting and differentiating venom proteins from hemorrhagic and neurotoxic snakes. A useful snakebite diagnostic guideline according to the lateral flow strip results and clinical symptoms was proposed to help physicians to use antivenoms appropriately. The two-test-line lateral flow strip assay could potentially be applied in an emergency room setting to help physicians diagnose and manage snakebite victims.

Proteomic characterization of six Taiwanese snake venoms: Identification of species-specific proteins and development of a SISCAPA-MRM assay for cobra venom factors.

Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Daboia russelii siamensis, Bungarus multicinctus and Naja atra are the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC-MS/MS and the total venom proteome was characterized by in-solution digestion coupled with LC-MS/MS. A total of 27-52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found in Viperidae species (D. acutus, T. stejnegeri, P. mucrosquamatus and D. russelii) were C-type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three-finger toxin and phospholipase A2 were the major components detected in the venom of Elapidae snakes (B. multicinctus and N. atra). This study also provided the first demonstration of some low-abundance proteins in these six snake venoms, including 5'-nucleotidase, glutaminyl-peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra-specific protein. We produced anti-peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA-MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1 µg crude venom proteins from three Naja species and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16-10.03 mg/g of venom protein). BIOLOGICAL SIGNIFICANCE: There are six medically significant venomous snakes in Taiwan. The venoms of the four Viperidae species (Deinagkistrodon acutus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus and Daboia russelii siamensis) cause local tissue swelling; this symptom is also seen in N. atra envenomation in humans, potentially complicating the differential diagnosis of envenomation by N. atra and Viperidae species. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species-specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species-specific venom protein(s) and new type(s) of antivenom.

Integrated omics profiling identifies hypoxia-regulated genes in HCT116 colon cancer cells.

Hypoxia is associated with poor prognosis in most solid tumors due to its multiple effects on therapy resistance, angiogenesis, apoptotic resistance, and tumor invasion/metastasis. Here we used a comprehensive omics profiling to investigate hypoxia-regulated gene expression in HCT116 colon cancer cells. Quantitative analyses of proteome and secretome were performed in HCT116 cells cultured under hypoxic or normoxic conditions. A total of 5700 proteins were quantified in proteome analysis and 722 proteins were quantified in secretome analysis. Both datasets were combined with the transcriptome and translatome datasets for further analysis. Verification of candidate proteins/genes in this integrated omics analysis was performed using immunoblotting and quantitative real-time RT-PCR analyses. We also performed polysome profiling to assess changes in translational efficiency of hypoxia-induced genes. Notably, several genes were differently regulated at the transcriptional and translational levels in HCT116 cells during hypoxia. Bioinformatics analysis suggested that hypoxia regulates translation of genes involved in extracellular matrix organization, extracellular exosomes, and protein processing in endoplasmic reticulum. Aberrations in these metabolic pathways appear to be correlated with an increased risk of tumor invasion/metastasis. BIOLOGICAL SIGNIFICANCE: This study integrates transcriptome/translatome and proteome/secretome to analyze gene expression changes in human colon cancer cells under hypoxic conditions. Candidate proteins/genes in this integrated omics analysis were further validated by immunoblotting, quantitative real-time RT-PCR, and polysome profiling. The datasets would be useful to uncover the molecular mechanisms of hypoxia-induced gene regulation in colorectal cancer.

Analysis of the efficacy of Taiwanese freeze-dried neurotoxic antivenom against Naja kaouthia, Naja siamensis and Ophiophagus hannah through proteomics and animal model approaches.

In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom-freeze-dried neurotoxic antivenom (FNAV)-against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two 'true' cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites.

Formation and characteristics of biomimetic mineralo-organic particles in natural surface water.

Recent studies have shown that nanoparticles exist in environmental water but the formation, characteristics and fate of such particles remain incompletely understood. We show here that surface water obtained from various sources (ocean, hot springs, and soil) produces mineralo-organic particles that gradually increase in size and number during incubation. Seawater produces mineralo-organic particles following several cycles of filtration and incubation, indicating that this water possesses high particle-seeding potential. Electron microscopy observations reveal round, bacteria-like mineral particles with diameters of 20 to 800 nm, which may coalesce and aggregate to form mineralized biofilm-like structures. Chemical analysis of the particles shows the presence of a wide range of chemical elements that form mixed mineral phases dominated by calcium and iron sulfates, silicon and aluminum oxides, sodium carbonate, and iron sulfide. Proteomic analysis indicates that the particles bind to proteins of bacterial, plant and animal origins. When observed under dark-field microscopy, mineral particles derived from soil-water show biomimetic morphologies, including large, round structures similar to cells undergoing division. These findings have important implications not only for the recognition of biosignatures and fossils of small microorganisms in the environment but also for the geochemical cycling of elements, ions and organic matter in surface water.