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Cyanobacteria bioactive compounds are chemical treasure troves for product discovery and development. The wound healing effects and antioxidant capacities of water extracts from Nostoc NIES-2111_MUM004 were evaluated via in vitro wound scratch assay and three antioxidant assays respectively. Results showed that the water extracts were protein-rich and exhibited good antioxidant properties in ABTS radical scavenging (11.27 ± 0.205 mg TAE g−1 extract), Ferric reducing antioxidant power (1652.71 ± 110.71 mg TAE g−1 extract) and β-carotene bleaching assay (354.90 ± 31.80 mg TAE g−1 extract). Also, extracts were non-cytotoxic in concentrations up to 250 µg/mL as reflected in cytotoxicity assay. Importantly, water extracts showed considerable proliferation and migration activity at 125 µg/mL with wound closure rate as high as 42.67%. Statistical correlation revealed no significant relationship (p > 0.05) between protein fraction and the wound healing properties, confirming that phycobiliproteins were not solely responsible for wound healing activities. Subsequent Q-TOF-LCMS analysis identified six protein families involved in enhancing the proliferation and migration of epithelial cells. These findings are antecedent in the uncovering of continuous supplies of bioactive compounds from new and sustainable sources. Ultimately, enriching the microalgae menu for applications in pharmaceutical, nutraceutical and cosmeceuticals.

Cytotoxin (CTX) is a three-finger toxin presents predominantly in cobra venom. The functional site of the toxin is located at its three hydrophobic loop tips. Its actual mechanism of cytotoxicity remains inconclusive as few conflicting hypotheses have been proposed in addition to direct cytolytic effects. The present work investigated the interaction between CTX and death receptor families via ensemble-based molecular docking and fluorescence titration analysis. Multiple sequence alignments of different CTX isoforms obtained a conserved CTX sequence. The three-dimensional structure of the conserved CTX was later determined using homology modelling, and its quality was validated. Ensemble-based molecular docking of CTX was performed with different death receptors, such as Fas-ligand and tumor necrosis factor receptor families. Our results showed that tumor necrosis factor receptor 1 (TNFR1) was the best receptor interacting with CTX attributed to the interaction of all three functional loops and evinced with low HADDOCK, Z-score and RMSD value. The interaction between CTX and TNFR1 was also supported by a concentration-dependent reduction of fluorescence intensity with increasing binding affinity. The possible intermolecular interactions between CTX and TNFR1 were Van der Waals forces and hydrogen bonding. Our findings suggest a possibility that CTX triggers apoptosis cell death through non-covalent interactions with TNFR1. Communicated by Ramaswamy H. Sarma

Naja sumatrana venom cytotoxin (sumaCTX) is a basic protein which belongs to three-finger toxin family. It has been shown to induce caspase-dependent, mitochondrial-mediated apoptosis in MCF-7 cells at lower concentrations. This study aimed to investigate the alteration of secretome in MCF-7 cells following membrane permeabilization by high concentrations of sumaCTX, using label-free quantitative (LFQ) approach. The degree of membrane permeabilization of sumaCTX was determined by lactate dehydrogenase (LDH) assay and calcein-propidium iodide (PI) assays. LDH and calcein-PI assays revealed time-dependent membrane permeabilization within a narrow concentration range. However, as toxin concentrations increased, prolonged exposure of MCF-7 cells to sumaCTX did not promote the progression of membrane permeabilization. The secretome analyses showed that membrane permeabilization was an event preceding the release of intracellular proteins. Bioinformatics analyses of the LFQ secretome revealed the presence of 105 significantly distinguished proteins involved in metabolism, structural supports, inflammatory responses, and necroptosis in MCF-7 cells treated with 29.8 μg/mL of sumaCTX. Necroptosis was presumably an initial stress response in MCF-7 cells when exposed to high sumaCTX concentration. Collectively, sumaCTX-induced the loss of membrane integrity in a concentration-dependent manner, whereby the cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis with increasing toxin concentrations.

Experiential learning is compromised in meeting the educational demands of our students during the challenging time of the COVID-19 pandemic. A more inclusive, flexible, and objective-oriented experiential learning environment is required. In this context, module-based experiential learning that is executable on a digital platform was designed. The learning module focused on protein biochemistry, contained a combination of asynchronous and synchronous activities categorized into 'Knowledge Hub' and 'Lab-based Movie', across 5 weeks. Digital and module-based experiential learning provides equitable, inclusive, and flexible access to students at remote locations. Furthermore, it is an objective-oriented and highly organized experiential learning framework that encourages students to engage and participate more in the learning process.

The phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are two major enzymes found in the venoms from most snake species. These enzymes have been structurally and functionally characterised for their pharmacological activities. Both PLA2 and LAAO from different venoms demonstrate considerable cytotoxic effects on cancer cells via induction of apoptosis, cell cycle arrest and suppression of proliferation. These enzymes produce more pronounced cytotoxic effects in cancer cells than normal cells, thus they can be potential sources as chemotherapeutic agents. It is proposed that PLA2 and LAAO contribute to an elevated oxidative stress due to their catalytic actions, for instance, the ability of PLA2 to produce reactive oxygen species during lipolysis and formation of H2O2 from LAAO catalytic activity which consequently lead to cell death. Nonetheless, the cell-death signalling pathways associated with exposure to these enzymatic toxins are not fully elucidated yet. Here in this review, we will discuss the cytotoxic effects of PLA2 and LAAO in relationship to their catalytic mechanisms and the underlying mechanisms of cytotoxic actions.

Cobra venom cytotoxin (CTX) is a non-enzymatic three-finger toxin that constitutes 40-60% of cobra venom. Thus, it plays an important role in the pathophysiology of cobra envenomation, especially in local dermonecrosis. The three-finger hydrophobic loops of CTX determine the cytotoxicity. Nevertheless, the actual mechanisms of cytotoxicity are not fully elucidated as they involve not only cytolytic actions but also intracellular signalling-mediated cell death pathways. Furthermore, the possible transition cell death pattern remains to be explored. The actual molecular mechanisms require further studies to unveil the relationship between different CTXs from different cobra species and cell types which may result in differential cell death patterns. Here, we discuss the biophysical interaction of CTX with the cell membrane involving four binding modes: electrostatic interaction, hydrophobic partitioning, isotropic phase, and oligomerisation. Oligomerisation of CTX causes pore formation in the membrane lipid bilayer. Additionally, the CTX-induced apoptotic pathway can be executed via death receptor-mediated extrinsic pathways and mitochondrial-mediated intrinsic pathways. We also discuss lysosomal-mediated necrosis and the occurrence of necroptosis following CTX action. Collectively, we provided an insight into concentration-dependent transition of cell death pattern which involves different mechanistic actions. This contributes a new direction for further investigation of cytotoxic pathways activated by the CTXs for future development of biotherapeutics targeting pathological effects caused by CTX.

Introduction There is growing evidence to suggest the importance of self-regulatory practices amongst older adults to sustain mobility. However, the decision to self-regulate driving is a complex interplay between an individual's preference and the influence of their social networks including spouse. To our best knowledge, the influence of an older adult's spouse on their decisions during driving transition has not been explored. Materials and methods This qualitative descriptive study was conducted amongst married older adults aged 60 years and above. All interview responses were transcribed verbatim and examined using thematic approach and interpretative description method. Results A total of 11 married couples were interviewed. Three major themes emerged: [1] Our roles in driving; [2] Challenges to continue driving; and, [3] Our driving strategies to ensure continued driving. Older couples adopted driving strategies and regulated their driving patterns to ensure they continued to drive safely. Male partners often took the active driving role as the principal drivers, while the females adopted a more passive role, including being the passenger to accompany the principal drivers or becoming the co-driver to help in navigation. Other coping strategies include sharing the driving duties as well as using public transportation or mixed mode transportation. Discussion Our findings suggest spouse play a significant role in their partners' decision to self-regulate driving. This underscores a need to recognise the importance of interdependency amongst couples and its impact on their driving decisions and outcomes.

The mechanism of suma-cytotoxin (sumaCTX)-induced cytotoxicity is poorly understood, especially in the transition of cell death pattern. This study examines its cytotoxicity, caspase-3/7 activity, mitochondrial functions, and occurrence of apoptosis. The sumaCTX triggered cytotoxicity in MCF-7 cells before 24 h, with activation of caspase-3/7 and mitochondrial depolarization, followed by a substantial percentage of AV+ apoptotic cells. The absence of HMGB1 suggested apoptosis was the profound cell death independent of its membrane perturbing effect, without transitioning into necrosis. Its cytotoxicity was a concentration-dependent but not time-dependent process. The findings provide valuable insight into mechanisms of sumaCTX-induced cytotoxicity in breast cancer cells.

The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia was investigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein families were detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipase A2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms of New World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteome also revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reported high level of adenosine in the venom. Also detected were 5′-nucleotidase (0.3%), hyaluronidase (0.1%) and cysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2, phosphodiesterase, 5′-nucleotidase and acetylcholinesterase activities but not l -amino acid oxidase activity. The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50 = 62.14 ± 0.87 μg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic action with phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent antivenom from Taiwan. Together, the findings provide insights into the composition and functions of the venom of this exotic oriental elapid snake. Biological significance While venoms of the New World coral snake have been extensively studied, literature pertaining to the Old World or Asiatic coral snake venoms remains lacking. This could be partly due to the inaccessibility to the venom of this rare species and infrequent cases of envenomation reported. This study identified and profiled the venom proteome of the Malayan blue coral snake (C. b. flaviceps) through SDS-PAGE and a high-resolution nano-LCMS/MS method, detailing the types and abundance of proteins found in the venom. The biological and toxic activities of the venom were also investigated, offering functional correlation to the venom proteome studied. Of note, the venom contains a unique toxin profile predominated with phospholipase A2 and cytotoxin with no detectable post-synaptic neurotoxin. The venom is moderately lethal to mice and the fatal effect could be cross-neutralized by a heterologous elapid bivalent antivenom from Taiwan. The findings enrich snake toxin databases and provide insights into the composition and pathogenesis of the venom of this exotic species.

Type II diabetes mellitus (T2DM) is a chronic non-communicable disease due to abnormal insulin actions causing uncontrolled hyperglycaemia. The treatment for T2DM, for instance, metformin and incretin mimetic, mainly focuses on the restoration of insulin sensitivity and secretion. Exendin-4 is a short incretin-mimetic peptide consisting of 39 amino acids. It is discovered in the venom of Heloderma suspectum as a full agonist for the glucagon-like peptide 1 (GLP-1) receptor and produces insulinotropic effects. It is more resistant to enzymatic degradation by dipeptidyl-peptidase-4 and has a longer half-life than the endogenous GLP-1; thus, it is further developed as an incretin hormone analogue used to treat T2DM. The helical region of the peptide first interacts with the extracellular N-terminal domain (NTD) of GLP-1 receptor while the C-terminal extension containing the tryptophan cage further enhances its binding affinity. After binding to the NTD of the receptor, it may cause the receptor to switch from its auto-inhibited state of the receptor to its auto-activated state. Exendin-4 enhances the physiological functions of β-cells and the up-regulation of GLP-1 receptors, thus reducing the plasma glucose levels. Moreover, exendin-4 has also been found to ameliorate neuropathy, nephropathy and ventricular remodelling. The therapeutic effects of exendin-4 have also been extrapolated into several clinical trials. Although exendin-4 has a reasonable subcutaneous bioavailability, its half-life is rather short. Therefore, several modifications have been undertaken to improve its pharmacokinetics and insulinotropic potency. This review focuses on the pharmacology of exendin-4 and the structure-function relationships of exendin-4 with GLP-1 receptor. The review also highlights some challenges and future directions in the improvement of exendin-4 as an anti-diabetic drug.

The venom proteome (venomics) of the Sri Lankan Daboia russelii was elucidated using 1D SDS PAGE nano-ESI-LCMS/MS shotgun proteomics. A total of 41 different venom proteins belonging to 11 different protein families were identified. The four main protein families are phospholipase A 2 (PLA 2 , 35.0%), snaclec (SCL, 22.4%, mainly platelet aggregation inhibitors), snake venom serine proteinase (SVSP, 16.0%, mainly Factor V activating enzyme) and snake venom metalloproteinase (SVMP, 6.9%, mainly heavy chain of Factor X activating enzyme). Other protein families that account for more than 1% of the venom protein include l -amino acid oxidase (LAAO, 5.2%), Kunitz-type serine proteinase inhibitor (KSPI, 4.6%), venom nerve growth factor (VNGF. 3.5%), 5′-nucleotidase (5′NUC, 3.0%), cysteine-rich secretory protein (CRISP, 2.0%) and phosphodiesterase (PDE, 1.3%). The venom proteome is consistent with the enzymatic and toxic activities of the venom, and it correlates with the clinical manifestations of Sri Lankan D . russelii envenomation which include hemorrhage, coagulopathy, renal failure, neuro-myotoxicity and intravascular hemolysis. The venom exhibited remarkable presypnatic neurotoxicity presumably due to the action of basic PLA 2 in high abundance (35.0%). Besides, SCLs, Factor X activating enzymes (SVMPs), SVSPs, and LAAOs are potential hemotoxins (50.5%), contributing to coagulopathy and hemorrhagic syndrome in Sri Lankan D . russelii envenomation. Significance The study demonstrated the proteomic profile of the Sri Lankan Russell's viper venom, unraveling its complex composition of toxins and correlations with major toxic activities. The types, numbers, and relative abundances of toxins were reported. The venom content was dominated by the neurotoxic basic phospholipases A 2 (> 30% of total protein abundance) and several hemotoxic or coagulopathic protein families (approximately 50% in total). The proteome correlates with the functional and toxinological characterizations of the venom, and reflects the pathophysiological effects of envenomation by the Sri Lankan Russell's viper. The venom proteomics may serve to propel the understanding on pathogenesis and treatment strategy for envenomation by this viper in Sri Lanka. The enriched database contributed by the proteomic findings will be useful for comparing venom variations among Russell's vipers from different geographical areas.

The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6-27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.

Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD50) (0.56–0.63 μg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A2, serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3–5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.

The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV, F(ab')2 ] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab')2 and venom antigens, as the terminal half-life and volume of distribution of the F(ab')2 in serum were comparable to that of venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity. The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses as clinically indicated in the presence of rebound antigenemia and symptom recurrence.

Background The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. Principal findings The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole venom (Tmax = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m. = 81.5%) compared to that of the phospholipase A2 (Fi.m. = 68.6%) or cardiotoxin (Fi.m. = 45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. Conclusion/Significance Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming., Author Summary Naja sumatrana is a medically important cobra species in Southeast Asia. The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition, its biological activities, as well as its pharmacokinetics. The present study on the pharmacokinetics of N. sumatrana venom shows that the systemic bioavailability of this venom in experimental envenomation is similar to N. sputatrix venom determined in an earlier study. The neurotoxin and cardiotoxin exhibited a more rapid absorption and elimination compared to the phospholipase A2 and the whole venom. The venom neurotoxin produced a higher systemic bioavailability than the cardiotoxin and phospholipase A2, suggesting that the neurotoxin plays the major toxic role in cobra bites.

Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms. The toxicokinetics of Naja sputatrix (Javan spitting cobra) venom was investigated following intravenous and intramuscular injections of the venom into rabbits using double-sandwich ELISA. The toxicokinetics of the venom injected intravenously fitted a two-compartment model. When the venom was injected intramuscularly, the serum concentration-time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were not significantly different (p 0.05) from that of the venom injected intravenously. The systemic bioavailability of the venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some cobra venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn't significantly alter the toxicokinetics of N. sputatrix venom although it significantly reduces the systemic bioavailability of the venom.

The lethal and enzymatic activities of venom from Naja sumatrana (Equatorial spitting cobra) were determined and compared to venoms from three other Southeast Asian cobras (Naja sputatrix, Naja siamensis and Naja kaouthia). All four venoms exhibited the common characteristic enzymatic activities of Asiatic cobra venoms: low protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase activities, moderately high acetylcholinesterase and hyaluronidase activities and high phospholipase A2. Fractionation of N. sumatrana venom by Resource® S cation exchange chromatography (GE Healthcare, USA) yielded nine major protein peaks, with all except the acidic protein peak being lethal to mice. Most of the protein peaks exhibit enzymatic activities, and L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, 5'-nucleotidase and hyaluronidase exist in multiple forms. Comparison of the Resource® S chromatograms of the four cobra venoms clearly indicates that the protein composition of N. sumatrana venom is distinct from venoms of the other two spitting cobras, N. sputatrix (Javan spitting cobra) and N. siamensis (Indochinese spitting cobra). The results support the revised systematics of the Asiatic cobra based on multivariate analysis of morphological characters. The three spitting cobra venoms exhibit two common features: the presence of basic, potentially pharmacologically active phospholipases A2 and a high content of polypeptide cardiotoxin, suggesting that the pathophysiological actions of the three spitting cobra venoms may be similar.