Abstract Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species’ venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation. Background Colombia coexist lifethreatening life threatening toxicity Methods Here C T n sp aff phospholipases hyaluronidases Results Conclusion envenomation

Abstract Background: Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. Methods: In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Results: Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Conclusion: Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research. Background dogs oncology malignancies women approaches terrificus rattlesnake 6 uses antitumor antiviral action Crotoxin βneurotoxin β neurotoxin CBPLA2 CBPLA CB PLA2 PLA (CB-PLA2 crotapotin, , (crotapotin) cancers Methods drug choice lines Results CBPLA2, PLA2, dosedependent dose dependent manner effect compounds apoptosis addition cells Conclusion Therefore CB-PLA activity inhibition proapoptotic pro apoptotic research (CB-PLA (crotapotin

Abstract Nerve injuries present a substantial challenge within the medical domain due to their prevalent occurrence and significant impact. In nerve injuries, a range of physiopathological and metabolic responses come into play to stabilize and repair the resulting damage. A critical concern arises from the disruption of connections at neuromuscular junctions, leading to profound degeneration and substantial loss of muscle function, thereby hampering motor tasks. While end-to-end neurorrhaphy serves as the established technique for treating peripheral nerve injuries, achieving comprehensive morphofunctional recovery remains a formidable challenge. In pursuit of enhancing the repair process, alternative and supportive methods are being explored. A promising candidate is the utilization of heterologous fibrin biopolymer, a sealant devoid of human blood components. Notably, this biopolymer has showcased its prowess in establishing a stable and protective microenvironment at the site of use in multiple scenarios of regenerative medicine. Hence, this scoping review is directed towards assessing the effects of associating heterologous fibrin biopolymer with neurorrhaphy to treat nerve injuries, drawing upon findings from prior studies disseminated through PubMed/MEDLINE, Scopus, and Web of Science databases. Further discourse delves into the intricacies of the biology of neuromuscular junctions, nerve injury pathophysiology, and the broader utilization of fibrin sealants in conjunction with sutures for nerve reconstruction procedures. The association of the heterologous fibrin biopolymer with neurorrhaphy emerges as a potential avenue for surmounting the limitations associated with traditional sealants while also mitigating degeneration in nerves, muscles, and NMJs post-injury, thereby fostering a more conducive environment for subsequent regeneration. Indeed, queries arise regarding the long-term regenerative potential of this approach and its applicability in reconstructive surgeries for human nerve injuries. impact damage junctions function tasks endtoend end process explored components Notably medicine Hence PubMedMEDLINE PubMed MEDLINE PubMed/MEDLINE Scopus databases pathophysiology procedures nerves muscles postinjury, postinjury post injury, post-injury regeneration Indeed longterm long term

Abstract Background: The relationship between viral infections and host factors holds high hopes for identifying the role of Interferon Lambda 3 (IFNL3) and Interleukin 6 (IL-6) polymorphisms in the development of Chronic Liver Disease (CLD) in patients infected with hepatitis Delta virus (HDV) in the Western Brazilian Amazon. Methods: Cross-sectional study conducted with a cohort of 40 chronic HDV patients, 27 with CLD and 13 without evident liver damage. Biological samples from the participants were analyzed using the polymerase chain reaction (PCR) technique, followed by sequencing by the automated Sanger method. Results: The rs8099917 T allele, from the IFNL3 gene, showed a higher frequency in both groups; however, it was not possible to establish an association with HDV infection [OR = 1.42 (0.42 - 4.75; p = 0.556 (95% CI). For IL-6, the rs1800795 G allele was superior to rs1800795 C. Analyzing both distributions in the studied groups, any association with HDV was absent (p > 0.05). Conclusion: The results suggest that the rs8099917 T/G (IFNL3) and rs1800795 G/C (IL-6) polymorphisms are not associated with the evolution of HDV in the studied population. Background IFNL (IFNL3 IL6 IL (IL-6 (CLD (HDV Amazon Methods Crosssectional Cross sectional 4 2 1 damage PCR (PCR technique method Results rs rs809991 gene groups however OR 142 42 1.4 0.42 042 0 (0.4 4.75 475 75 0556 556 0.55 95% 95 (95 CI. CI . CI) IL6, 6, IL-6 rs180079 C 0.05. 005 0.05 05 0.05) Conclusion TG GC population (IFNL (IL- rs80999 14 1. 0.4 04 (0. 4.7 47 7 055 55 0.5 9 (9 IL- rs18007 00 0.0 (IL rs8099 0. (0 4. 5 ( rs1800 rs809 rs180 rs80 rs18 rs8 rs1

Abstract Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and β), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil. Brazil fatalities modality effects T scorpions reproduction α β, β , β) channels Nevertheless metalloproteases proteases phospholipases hyaluronidases strategies

Abstract Background: The bioactive peptides derived from snake venoms of the Viperidae family species have been promising as therapeutic candidates for neuroprotection due to their ability to prevent neuronal cell loss, injury, and death. Therefore, this study aimed to evaluate the cytoprotective effects of a synthetic proline-rich oligopeptide 7a (PRO-7a; <EDGPIPP) from Bothrops jararaca snake, on oxidative stress-induced toxicity in neuronal PC12 cells and astrocyte-like C6 cells. Methods: Both cells were pre-treated for four hours with different concentrations of PRO-7a, submitted to H2O2-induced damage for 20 h, and then the oxidative stress markers were analyzed. Also, two independent neuroprotective mechanisms were investigated: a) L-arginine metabolite generation via argininosuccinate synthetase (AsS) activity regulation to produce agmatine or polyamines with neuroprotective properties; b) M1 mAChR receptor subtype activation pathway to reduce oxidative stress and neuron injury. Results: PRO-7a was not cytoprotective in C6 cells, but potentiated the H2O2-induced damage to cell integrity at a concentration lower than 0.38 μM. However, PRO-7a at 1.56 µM, on the other hand, modified H2O2-induced toxicity in PC12 cells by restoring cell integrity, mitochondrial metabolism, ROS generation, and arginase indirect activity. The α-Methyl-DL-aspartic acid (MDLA) and L-NΩ-Nitroarginine methyl ester (L-Name), specific inhibitors of AsS and nitric oxide synthase (NOS), which catalyzes the synthesis of polyamines and NO from L-arginine, did not suppress PRO-7a-mediated cytoprotection against oxidative stress. It suggested that its mechanism is independent of the production of L-arginine metabolites with neuroprotective properties by increased AsS activity. On the other hand, the neuroprotective effect of PRO-7a was blocked in the presence of dicyclomine hydrochloride (DCH), an M1 mAChR antagonist. Conclusions: For the first time, this work provides evidence that PRO-7a-induced neuroprotection seems to be mediated through M1 mAChR activation in PC12 cells, which reduces oxidative stress independently of AsS activity and L-arginine bioavailability. Background loss injury death Therefore prolinerich proline rich PRO7a PROa PRO (PRO-7a <EDGPIPP EDGPIPP stressinduced induced PC PC1 astrocytelike astrocyte like C Methods pretreated pre treated PRO7a, 7a, H2O2induced HOinduced H2O2 H O 2 h analyzed Also investigated Larginine L arginine (AsS b M Results 038 0 38 0.3 μM However 156 1 56 1.5 µM hand metabolism αMethylDLaspartic α Methyl DL aspartic MDLA (MDLA LNΩNitroarginine NΩ Nitroarginine LName, LName Name , (L-Name) NOS, NOS (NOS) Larginine, arginine, PRO7amediated PROamediated DCH, DCH (DCH) antagonist Conclusions time PRO7ainduced PROainduced bioavailability HO H2O 03 3 0. 15 5 1. (L-Name (NOS amediated (DCH ainduced

Abstract Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species. Background Simon 189 Trechaleidae uncharacterized Medina 2006 Keyserling, Keyserling (Keyserling 1878, 1878 , 1878) Methods total 8 amplified Americas distances C electrostimulation HPLC SDSPAGE, SDSPAGE SDS PAGE, PAGE SDS-PAGE insects Results 94 9497% 9497 97% 97 94-97 conspecifics crickets flies components ChiaTx ChiaTx2 Conclusion COIbased based 18 200 187 9 949 94-9 1 20 94- 2

Abstract Background: Loxoscelism refers to a set of clinical manifestations caused by the bite of spiders from the Loxosceles genus. The classic clinical symptoms are characterized by an intense inflammatory reaction at the bite site followed by local necrosis and can be classified as cutaneous loxoscelism. This cutaneous form presents difficult healing, and the proposed treatments are not specific or effective. This study aimed to evaluate the protective effect of mesenchymal stromal cells-derived secretome on dermonecrosis induced by Loxosceles intermedia spider venom in rabbits. Methods: Sixteen rabbits were distributed into four groups (n = 4). Except for group 1 (G1), which received only PBS, the other three groups (G2, G3, and G4) were initially challenged with 10 μg of L. intermedia venom, diluted in 100 μL of NaCl 0.9%, by intradermic injection in the interscapular region. Thirty minutes after the challenge all groups were treated with secretome, except for group 2. Group 1 (G1-control group) received intradermal injection (ID) of 60 μg of secretome in 0.15 M PBS; Group 2 (G2) received 0.9% NaCl via ID; Group 3 (G3) received 60 μg of secretome, via ID and Group 4 (G4), received 60 μg of secretome by intravenous route. Rabbits were evaluated daily and after 15 days were euthanized, necropsied and skin samples around the necrotic lesions were collected for histological analysis. Results: Rabbits of G1 did not present edema, erythema, hemorrhagic halo, or necrosis. In animals from G2, G3, and G4, edema appeared after 6h. However, minor edema was observed in the animals of G2 and G3. Hemorrhagic halo was observed in animals, six hours and three days after, on G2, G3, and G4. Macroscopically, in G4, only one animal out of four had a lesion that evolved into a dermonecrotic wound. No changes were observed in the skin of the animals of G1, by microscopic evaluation. All animals challenged with L. intermedia venom showed similar alterations, such as necrosis and heterophilic infiltration. However, animals from G4 showed fibroblast activation, early development of connective tissue, neovascularization, and tissue re-epithelialization, indicating a more prominent healing process. Conclusion: These results suggest that secretome from mesenchymal stromal cells cultured in a xeno-free and human component-free culture media can be promising to treat dermonecrosis caused after Loxosceles spiders bite envenoming. Background genus loxoscelism effective cellsderived derived Methods n 4. . 4) G , (G1) PBS (G2 G3 L 09 0 9 region G1control Gcontrol control (ID 6 015 0.1 0.9 (G3 (G4) route euthanized analysis Results erythema 6h h However Macroscopically wound evaluation alterations infiltration activation neovascularization reepithelialization, reepithelialization re epithelialization, epithelialization re-epithelialization process Conclusion xenofree xeno free componentfree component envenoming (G1 (G 01 0. (G4

Abstract Background: Acid-sensing ion channel 1a (ASIC1a) plays a critical role in physiological and pathological processes. To further elucidate the biological functions of ASICs and their relationships with disease occurrence and development, it is advantageous to investigate and develop additional regulatory factors for ASICs. Methods: In this study, cation exchange chromatography was used to separate seven chromatographic components from Naja naja atra venom. Capillary electrophoresis was employed to detect that Ⅶ peak component containing a main protein Ⅶ-2, which could bind to ASIC1a. The analgesic effects of Ⅶ-2 protein were determined using hot plate methods, and ASIC1a expression in spinal cord tissue from rats with inflammatory pain was detected using western blot. Results: The purified Ⅶ-2 protein named Naja naja atra venom-Ⅶ-2 (NNAV-Ⅶ-2) was obtained by Sephadex G-50 gel filtration, which exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 6.7 kD. Remarkably, the NNAV-Ⅶ-2 protein demonstrated a significant analgesic effect and downregulated ASIC1a expression in the spinal cord tissue of rats with inflammatory pain. Conclusions: The analgesic mechanism of the NNAV-Ⅶ-2 protein may be associated with its binding to ASIC1a, consequently downregulating ASIC1a expression in neural tissues. Background Acidsensing Acid sensing ASICa ASIC (ASIC1a processes development Methods study venom Ⅶ2, Ⅶ2 2, 2 Ⅶ- methods blot Results venomⅦ2 venom-Ⅶ- NNAVⅦ2 NNAV (NNAV-Ⅶ-2 G50 G 50 G-5 filtration sulfatepolyacrylamide sulfate polyacrylamide 67 6 7 6. kD Remarkably NNAV-Ⅶ- Conclusions tissues venomⅦ venom-Ⅶ NNAVⅦ (NNAV-Ⅶ- G5 5 G- NNAV-Ⅶ venom- (NNAV-Ⅶ NNAV- (NNAV- (NNAV

Abstract Background: Paracoccidioidomycosis (PCM) is a severe granulomatous disease. The hallmark of this mycosis is fibrin degradation and granuloma formation as a result of a wound-healing process in the context of excessive inflammation. Therefore, as the content of collagen can be assessed by the methodology described in this manuscript, we propose that the content of hydroxyproline (HYP) be employed as a new and efficient measurement of granulomatous lesions developed. To estimate the level of HYP the major byproduct of the degradation process, we hypothesized that this simple and efficient technique could serve as a marker of disease severity. Methods: Five B10.A female mice were infected with P. brasiliensis and, after 15 days, the omentum was removed, subjected to histopathological analysis or processed (i.e. deproteinized and derivatized), and further analyzed on a reverse phase HPLC using a C-18 column. The omentum of five uninfected controls was also collected and similarly analyzed. Results: Infected mice showed numerous, disseminated paracoccidioidomycotic lesions, as well as marked collagen deposits, as observed in histopathologic analysis, and high levels of HYP. Normal uninfected mice showed no granulomas, little or no deposits of collagen fibers, and very low levels of HYP, as evaluated by HPLC. Our results show that the disease intensity as evaluated number and the morphology of the granulomatous lesions were correlated to the HYP levels using small tissue samples from the omentum, the main target organ of P. brasiliensis. Conclusions: Here we propose an alternative methodology to follow disease evolution and, to some extent, fungal load in experimental P. brasiliensis infection and suggest its usefulness to other diseases with pronounced fibrin degradation. Background PCM (PCM woundhealing wound healing inflammation Therefore manuscript (HYP developed severity Methods B10A BA B10 A B P 1 days removed i.e. ie i e (i.e derivatized, derivatized , derivatized) C18 C 18 C-1 column Results numerous granulomas fibers Conclusions extent B1 i.e C1 C-

Abstract The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: “electrophysiology”, “patch-clamp techniques”, “Ca2+ channels”, “K+ channels”, “cnidarian venoms”, “cone snail venoms”, “scorpion venoms”, “spider venoms”, “snake venoms”, “cardiac myocytes”, “dorsal root ganglia”, and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel’s structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins’ source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering. voltagegated voltage gated assays patchclamp patch clamp However origin approaches patchclamp. clamp. MEDLINEPubMed MEDLINE PubMed descriptors electrophysiology, electrophysiology , “electrophysiology” techniques, techniques” Ca2+ Ca2 Ca “Ca2 channels, channels” K+ K “K venoms, venoms venoms” scorpion spider snake cardiac myocytes, myocytes myocytes” dorsal ganglia, ganglia ganglia” review First channel s channelmodulating modulating e.g. eg e g (e.g snails scorpions spiders snakes, snakes snakes) Finally cells fields toxinology toxicology pharmacology products biophysics biomedicine bioengineering “electrophysiology “Ca e.g

Abstract Background: Phonotimpus pennimani (Araneae, Phrurolithidae) is a small-sized (3-5 mm) spider endemic to the Tacaná volcano in Chiapas, Mexico, where it is found in soil litter of cloud forests and coffee plantations. Its venom composition has so far not been investigated, partly because it is not a species of medical significance. However, it does have an important impact on the arthropod populations of its natural habitat. Methods: Specimens were collected in Southeastern Mexico (Chiapas) and identified taxonomically by morphological characteristics. A partial sequence from the mitochondrial gene coxI was amplified. Sequencing on the Illumina platform of a transcriptome library constructed from 12 adult specimens revealed 25 toxin or toxin-like genes. Transcripts were validated (RT-qPCR) by assessing the differential expression of the toxin-like PpenTox1 transcript and normalising with housekeeping genes. Results: Analysis of the coxI-gene revealed a similarity to other species of the family Phrurolithidae. Transcriptome analysis also revealed similarity with venom components of species from the families Ctenidae, Lycosidae, and Sicariidae. Expression of the toxin-like PpenTox1 gene was different for each developmental stage (juvenile or adult) and also for both sexes (female or male). Additionally, a partial sequence was obtained for the toxin-like PpenTox1 from DNA. Conclusion: Data from the amplification of the mitochondrial coxI gene confirmed that P. pennimani belongs to the family Phrurolithidae. New genes and transcripts coding for venom components were identified. Background Araneae, Araneae (Araneae Phrurolithidae smallsized small sized 35 3 5 (3- mm Chiapas plantations investigated significance However habitat Methods (Chiapas characteristics amplified 1 2 toxinlike like RTqPCR RT qPCR (RT-qPCR PpenTox Results coxIgene Ctenidae Lycosidae Sicariidae juvenile female male. male . male) Additionally DNA Conclusion P (3 (

Abstract The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies. 90 far processes cocktails slowmoving slow moving harsh environment venom identity diversity potential unexplored biodiversity 3 cancellatus regius villepinii characterized studied date studies 9

Abstract Background: Isoliquiritigenin (ISL) presents antitumor effects against melanoma cells. It is known that various circular RNAs (circRNAs) are involved in the development of melanoma. Therefore, the present study aims to investigate the molecular mechanisms of ISL and circ_0002860. Methods: Circ_0002860, microRNA-431-5p (miR-431-5p) and member RAS oncogene family (RAB9A) were detected through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. Cell viability was examined via cell counting kit-8 assay. The proliferation ability was assessed using colony formation assay. Cell apoptosis and cell cycle were determined by flow cytometry. Transwell assay was used for detection of migration and invasion. Western blot was conducted for protein analysis. Target binding was confirmed via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In vivo research was performed through xenograft tumor assay. Results: Circ_0002860 was downregulated by ISL in melanoma cells. ISL-induced inhibitory effects on cell proliferation, cell cycle progression, migration and invasion were alleviated by circ_0002860 overexpression. MiR-431-5p was a target of circ_0002860. Circ_0002860 eliminated the ISL-induced tumor inhibition via sponging miR-431-5p in melanoma cells. Circ_0002860 elevated the RAB9A level by targeting miR-431-5p. The function of ISL was related to miR-431-5p/RAB9A axis in melanoma progression. Tumor growth was reduced by ISL in vivo through downregulating circ_0002860 to regulate miR-431-5p and RAB9A levels. Conclusion: The current data indicates that ISL suppressed cell malignant progression of melanoma via targeting the circ_0002860/miR-431-5p/RAB9A pathway. Background (ISL cells circRNAs (circRNAs Therefore circ0002860 circ 0002860 Methods Circ0002860 Circ microRNA4315p microRNAp microRNA 431 5p p miR4315p miRp miR (miR-431-5p RABA RAB A (RAB9A transcriptionquantitative transcription quantitative RTqPCR RT qPCR (RT-qPCR kit8 kit 8 kit- cytometry analysis dualluciferase dual luciferase RIP (RIP Results Circ_000286 ISLinduced induced circ_000286 overexpression MiR4315p MiRp MiR miR4315p. 5p. miR4315p/RAB9A miR4315pRAB9A miRpRABA 5p/RAB9A levels Conclusion circ_0002860/miR4315p/RAB9A circ0002860miR4315pRAB9A circmiRpRABA circ_0002860/miR pathway circ000286 000286 Circ000286 43 Circ_00028 circ_00028 pRAB 5pRAB9A pRABA circ0002860miR circmiR circ00028 00028 Circ00028 4 Circ_0002 circ_0002 circ0002 0002 Circ0002 Circ_000 circ_000 circ000 000 Circ000 Circ_00 circ_00 circ00 00 Circ00 Circ_0 circ_0 circ0 0 Circ0 Circ_ circ_

Abstract 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. Background freezedried freeze dried FN (FN s Naja atra However envenomationrelated related snakebites Methods groups 1 gauzes fluids significance timing Results n 11 73%. 73 73% . 73%) 4 (4 IQR26 IQR 2 6 IQR:2- IQR12, IQR12 2, IQR:1-2 0.05, 005 0.05 , 0 05 0.05) beforeafter 0.0079. 00079 0.0079 0079 0.0079) symptoms longterm long term morbidity Conclusions 7 ( IQR2 IQR:2 IQR1 IQR:1- 00 0.0 0007 0.007 007 IQR: IQR:1 0. 000 0.00