Paracapillaria (Ophidiocapillaria) siamensis sp. nov. (Nematoda: Trichuroidea): a new nematode in Naja kaouthia from Thailand.

A comprehensive investigation, incorporating both morphological and molecular analyses, has unveiled the existence of a hitherto unknown nematode species, Paracapillaria (Ophidiocapillaria) siamensis sp. nov., residing in the intestine of the monocled cobra, Naja kaouthia, in the central region of Thailand. This study integrates morphological characteristics, morphometric examination, scanning electron microscopy and molecular phylogenetic analysis (COI, 18S rRNA and ITS1 genes). The findings place the newly described species within the subgenus Ophidiocapillaria, elucidating its distinctive characteristics, including a frame-like proximal spicule shape, approximate lengths of 19 000 and 22 500 µm with approximate widths of 90 and 130 µm for males and females, 39‒45 stichocytes, elevated lips without protrusion, a dorsal bacillary band stripe with an irregular pattern of bacillary cells and evidence of intestinal infection. These features serve to differentiate it from other species within the same subgenus, notably Paracapillaria (Ophidiocapillaria) najae De, , a species coexisting P. siamensis sp. nov. in the monocled cobra from the same locality. This study addresses the co-infection of the novel species and P. najae within the same snake host, marking the second documented instance of a paracapillariid species in the monocled cobra within the family Elapidae. The genetic characterization supports the formal recognition of P. siamensis sp. nov. as a distinct species, thereby underscoring its taxonomic differentiation within the Capillariidae family. This research identifies and characterizes the new nematode species, contributing valuable insights into the taxonomy of this nematode.

Redescription and new record of Paracapillaria (Ophidiocapillaria) najae (Nematoda: Trichuroidea) in the monocled cobra Naja kaouthia from central Thailand: morphological and molecular insights.

The parasitic nematode Paracapillaria (Ophidiocapillaria) najae De, 1998, found in the Indian cobra Naja naja is redescribed and re-illustrated in the present study. The monocled cobra Naja kaouthia was discovered to be a new host for this parasite in central Thailand. A comprehensive description extending the morphological and molecular characteristics of the parasites is provided to aid species recognition in future studies. The morphometric characters of 41 parasites collected from 5 cobra specimens are compared with those described in the original studies. Phylogenetic analyses using mitochondrial cytochrome c oxidase subunit 1 and nuclear 18S ribosomal RNA genes were performed to provide novel information on the systematics of P. najae. Similar characteristics were observed in the examined nematode samples, despite being found in different hosts, confirming their identity as P. najae. The molecular genetic results support the species status of P. najae, indicating P. najae is well defined and separated from other related nematode species in the family Capillariidae. Morphological descriptions, genetic sequences, evolutionary relationships among capillariids and new host and distribution records of P. najae are discussed. Paracapillaria najae specimens found in the Thai cobra had some morphological variation, and sexual size dimorphism was also indicated. Paracapillaria najae was found to infect various cobra host species and appeared to be common throughout the Oriental regions, consistent with its hosts' distribution.

Genome-wide SNP analysis of Siamese cobra (Naja kaouthia) reveals the molecular basis of transitions between Z and W sex chromosomes and supports the presence of an ancestral super-sex chromosome in amniotes.

Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to transposable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors.

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent "a hypothetical ancestral super-sex chromosome" or random distribution?

BACKGROUND: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. RESULTS: We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. CONCLUSIONS: The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes.

Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion­deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.

Acute phase reactions in Daboia siamensis venom and fraction-induced acute kidney injury: the role of oxidative stress and inflammatory pathways in in vivo rabbit and ex vivo rabbit kidney models.

Background: This study examines the direct nephrotoxic effects of Daboia siamensis venom (RVV) and venom fractions in in vivo and isolated perfused kidneys (IPK) to understand the role of inflammation pathways and susceptibility to oxidative stress in venom or fraction-induced acute renal failure. Methods: We administered RVV and its venom fractions (PLA2, MP, LAAO, and PDE) to rabbits in vivo and in the IPK model. We measured oxidative stress biomarkers (SOD, CAT, GSH, and MDA) in kidney tissue, as well as inflammatory cytokines (TNF-α, IL-1ß, IFN-γ, IL-4, IL-5, and IL-10), MDA and GSH levels in plasma and urine. We also calculated fractional excretion (FE) for pro-/anti-inflammatory cytokines and oxidative stress biomarkers, including the ratios of pro-/anti-inflammatory cytokines in urine after envenomation. Results: In both kidney models, significant increases in MDA, SOD, CAT, and GSH levels were observed in kidney tissues, along with elevated concentrations of MDA and GSH in plasma and urine after injecting RVV and venom fractions. Moreover, RVV injections led to progressive increases in FEMDA and decreases in FEGSH. The concentrations of IL-4, IL-5, IL-10, IFN-γ, and TNF-α in plasma increased in vivo, as well as in the urine of the IPK model, but not for IL-1ß in both plasma and urine after RVV administrations. Urinary fractional excretion of TNF-α, IL-1ß, IFN-γ, IL-4, IL-5, and IL-10 tended to decrease in vivo but showed elevated levels in the IPK model. A single RVV injection in vivo disrupted the balance of urinary cytokines, significantly reducing either the TNF-α/IL-10 ratio or the IFN-γ/IL-10 ratio. Conclusion: RVV induces renal tubular toxicity by increasing oxidative stress production and elevating inflammatory cytokines in urine. During the acute phase of acute kidney injury, the balance of urine cytokines shifts toward anti-inflammatory dominance within the first two hours post-RVV and venom fractions.

Venom-gland transcriptomics of the Malayan pit viper (Calloselasma rhodostoma) for identification, classification, and characterization of venom proteins.

The Malayan pit viper (Calloselasma rhodostoma) is a hemotoxic snake widely found in Southeast Asia and is responsible for the majority of poisoning cases in this region, including Thailand. However, a comprehensive knowledge of the venom protein profile and classification, as well as novel venom proteins, of this viper is still limited. Recently, the detailed composition of several snake venoms has been discovered through the use of transcriptome analysis. Therefore, the aim of this study was to employ a next-generation sequencing platform and bioinformatics analysis to undertake venom-gland de novo transcriptomics of Malayan pit vipers. Furthermore, 21,272 functional coding genes were identified from 36,577 transcripts, of which 314 transcripts were identified as toxin proteins, accounting for 61.41% of total FPKM, which can be categorized into 22 toxin gene families. The most abundant are snake venom metalloproteinase kistomin (P0CB14) and zinc metalloproteinase/disintegrin (P30403), which account for 60.47% of total toxin FPKM and belong to the SVMP toxin family, followed by snake venom serine protease 1 (O13059) and Snaclec rhodocetin subunit beta (P81398), which account for 6.84% and 5.50% of total toxin FPKM and belong to the snake venom serine protease (SVSP) and Snaclec toxin family, respectively. Amino acid sequences of the aforementioned toxins were compared with those identified in other important medical hemotoxic snakes from Southeast Asia, including the Siamese Russell's viper (Daboia siamensis) and green pit viper (Trimeresurus albolabris), in order to analyze their protein homology. The results demonstrated that ranges of 58%-62%, 31%-60%, and 48%-59% identity was observed among the SVMP, Snaclec, and SVSP toxin families, respectively. Understanding the venom protein profile and classification is essential in interpreting clinical symptoms during human envenomation and developing potential therapeutic applications. Moreover, the variability of toxin families and amino acid sequences among related hemotoxic snakes found in this study suggests the use and development of universal antivenom for the treatment of envenomating patients is still challenging.

Comparative compositional and functional venomic profiles among venom specimens from juvenile, subadult and adult Russell's viper ( Daboia siamensis ): correlation with renal pathophysiology in experimental rabbits.

Background: Eastern Russell's viper (Daboia siamensis) is one of the most medically significant snakes responsible for the development of acute renal failure. However, variation of the clinical picture and renal pathophysiology following bites by young and adult D. siamensis have not been elucidated. Methods: In this study, we analyzed the venomic profiles of D. siamensis at different maturation stages of juvenile, subadult and adult groups. The same pooled venom from each group was subjected to enzymatic, electrophoretic and proteomic analysis, including sublethal toxicity (0.1 mg/kg iv.) examined on bodily functions by comparing the venom compositional and functional profiles among venom specimens from juvenile, subadult and adult D. siamensis by correlating them with the renal pathophysiology in experimental rabbits. Results: The comparative studies revealed that juvenile venom possessed higher phospholipase A2, metalloproteinase and serine proteinase levels, while subadult and adult venoms contained more L-amino acid oxidase, phosphodiesterase, the Kunitz-type serine protease inhibitor, disintegrin families and endothelial growth factor. An in vivo study revealed that the adult and subadult venoms caused persistent hypotension and bradycardia, while thrombocytopenia was a more characteristic effect of juvenile venom. All venom age groups showed significant reductions in renal hemodynamics and electrolyte excretions. The juvenile venom caused a higher tubulonephrosis lesion score than adult and subadult venoms. Conclusions: The D. siamensis venom shows an ontogenetic shift in its compositions and activities. Renal function alterations after envenomation depend on either the synergistic actions of different venom components or the disproportionate expression between the concentrations of enzymatic and non-enzymatic proteins in each age venom group. The high proportion of enzymatic toxin proteins in the juvenile venom results in greater nephrotoxicity.

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy.

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.

Combined proteomic strategies for in-depth venomic analysis of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand.

This study focuses on comprehensive characterization of the venom proteome of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand. H. schistosus can be considered as the deadliest sea snake commonly found in the Pacific and Indian oceans. Their envenomation causes muscular paralysis and rhabdomyolysis. To develop effective treatment for this snakebite, it is necessary to understand the detailed venom composition. In this study, multiple mass spectrometry-based approaches were employed. Bottom-up proteomics revealed that tryptic digestion in-solution provided a higher number of toxin proteins identified and a larger sequence coverage, compared to in-gel digestion. In addition, a venom gland transcriptome-derived database was constructed and used as a reference, which 43 known and novel toxin proteins were identified using this database and the UniProtKB. Three-finger toxin and phospholipase A2 were shown to be top two most abundant protein families. Minor compositions included other toxin families and a number of non-toxin proteins. Moreover, a hybrid de novo sequencing was performed to enhance identification of the small proteins/peptides. Using non-digested samples, there were 46 predicted toxin peptides. The finding from this study could lead to a better understanding in pathological effects of the snakebite and the future development of effective antivenoms. SIGNIFICANCE: This study provides a better understanding of the venom proteome composition of the beaked sea snake (H. schistosus) found in the Gulf of Thailand, using a combination of different sample preparation techniques, Serpentes protein database searching, transcriptome-derived protein database searching, and a hybrid de novo peptide sequencing strategy. It revealed 13 toxin protein families and novel proteins in the beaked sea snake venom including new species of phospholipase A2s (PLA2s) and three-finger toxins (3FTxs). It could serve as a basis for the development of snakebite treatments and for the discovery of novel pharmaceutical drugs from the toxin peptides.

Identification of Daboia siamensis venome using integrated multi-omics data.

Snakebite, classified by World Health Organization as a neglected tropical disease, causes more than 100,000 deaths and 2 million injuries per year. Currently, available antivenoms do not bind with strong specificity to target toxins, which means that severe complications can still occur despite treatment. Moreover, the cost of antivenom is expensive. Knowledge of venom compositions is fundamental for producing a specific antivenom that has high effectiveness, low side effects, and ease of manufacture. With advances in mass spectrometry techniques, venom proteomes can now be analyzed in great depth at high efficiency. However, these techniques require genomic and transcriptomic data for interpreting mass spectrometry data. This study aims to establish and incorporate genomics, transcriptomics, and proteomics data to study venomics of a venomous snake, Daboia siamensis. Multiple proteins that have not been reported as venom components of this snake such as hyaluronidase-1, phospholipase B, and waprin were discovered. Thus, multi-omics data are advantageous for venomics studies. These findings will be valuable not only for antivenom production but also for the development of novel therapeutics.

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

A new karst-dwelling, colorful pitviper (Viperidae: Trimeresurus) from northern Peninsular Thailand.

We describe a colorful and distinctively patterned, karst-dwelling pitviper, Trimeresurus kuiburi sp. nov., from the isolated, coastal massif of Khao Sam Roi Yot in Kui Buri District, Prachuap Khiri Khan Province, in northern Peninsular Thailand. The new species, member of the 'Cryptelytrops group' sensu Malhotra Thorpe (2004) and morphologically and genetically allied to Trimeresurus kanburiensis and T. venustus, differs from all pitviper taxa by a combination of red/purple bands on a green dorsum; a white concave suborbital stripe in males (straight and less visible in females); white, spaced vertebral dots in males (absent in females); pale green belly lacking dark dots or stripe on the lateral sides of the ventrals; partially fused first supralabial and nasal scale; 19 dorsal scale rows at midbody; 164171 ventrals; 6365 subcaudals in males, 5153 in females; maximal known SVL of 451 mm; and long, papillose hemipenes.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

Acute kidney injury (AKI) following Eastern Russell's viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

The pathophysiological effects of Russell's viper (Daboia siamensis) venom and its fractions in the isolated perfused rabbit kidney model: A potential role for platelet activating factor.

The pathophysiological effects of Russell's viper venom (RVV) and its fractions, including phospholipase A2 (RvPLA2), metalloprotease (RvMP), L-amino acid oxidase (RvLAAO), and phosphodiesterase (RvPDE) on renal functions were investigated using the isolated perfused rabbit kidney (IPK) model. Moreover, whether their effects on renal alterations were promoted by platelet activating factor (PAF) was tested using the PAF receptor antagonist, WEB 2086. There was a marked reduction in the perfusion pressure (PP) and renal vascular resistance (RVR) 10 min after RVV administration (1.0 mg/100 ml of perfusate), thereafter both PP and RVR gradually increased and approached the control level within 90 min. These effects were abolished by pretreatment with WEB2086 (2 µg/µl). Administration with RvPLA2 (280 µg/ml), RvMP (280 µg/ml), or RvLAAO (135 µg/ml) alone increased both the PP and RVR, whereas RvPDE (100 µg/ml) reduced both the PP and RVR. Pretreatment with WEB 2086 completely abolished the effects induced by RvMP, but not the other fractions. The RVV also caused a marked decrease in the glomerular filtration rate (GFR), urinary flow rate (UF), and osmolar clearance (Cosm), and these effects were not inhibited by pretreatment with WEB2086. Each RVV fraction also increased, to varying extents, the GFR, UF, and Cosm, and these effects induced by RvPLA2 or RvMP, but not the other fractions, were completely blocked by WEB 2086. Changes in percent filtered Na+ and K+ excreted in the IPK by RVV, RvPDE, and RvMP were abolished by pretreatment with WEB 2086. Histological evaluation profiled mainly tubulonephrosis in the treated kidney. These results reveal that the alterations in renal functions induced by RVV and its fractions are due to the synergistic action of the different components of snake venom, instead of the action of a single component. The effects of RVV and its fractions in rabbit IPK are mediated at least in part by PAF.

Acanthosaura aurantiacrista (Squamata: Agamidae), a new long horn lizard from northern Thailand.

BACKGROUND: In Thailand, five species of Acanthosaura have been recorded so far, including Acanthosaura armata from the southern region, A. cardamomensis from the eastern region, A. crucigera from the western region, A. lepidogaster from the northern region and A. phuketensis from the Phuket Island and south-western region. However, comprehensive studies of diversity patterns and distribution of Acanthosaura are still lacking in some areas and need further information for designating areas of special conservation importance and nature protection planning in Thailand. NEW INFORMATION: Acanthosaura aurantiacrista is a new species of long-horned lizard of the genus Acanthosaura from northern Thailand. It is distinguished from all other species of Acanthosaura by a dagger-like nuchal spine with yellowish-orange colouration in females, bright yellow colouration in males and a combination of other morphological characters: a greater tail length to snout-vent length ratio; a larger postorbital spine, nuchal spine, dorsal spine and occipital spine compared to its head length; a smaller diastema to snout-vent length ratio; a greater number of subdigital lamellae on the fourth finger and fourth toe; and a larger gular pouch than other Acanthosaura species. Analysis of mitochondrial ND2 gene sequences revealed a sister clade between the A. aurantiacrista lineage and the A. crucigera lineage with a 100% probability of divergence, according to Bayesian analysis and strong support value for Maximum Likelihood analysis. The pairwise distance ranged from 13.8-15.0% between A. aurantiacrista and A. cardamomensis, 10.9-14.5% between A. aurantiacrista and A. crucigera and 0-1.2% amongst A. aurantiacrista populations. The discovery of this lizard increases the known endemic herpetological diversity and underscores the importance of conservation in the mountain rainforest region of northern Thailand.

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni).

The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry-based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims.

Existence of Bov-B LINE Retrotransposons in Snake Lineages Reveals Recent Multiple Horizontal Gene Transfers with Copy Number Variation.

Transposable elements (TEs) are dynamic elements present in all eukaryotic genomes. They can "jump" and amplify within the genome and promote segmental genome rearrangements on both autosomes and sex chromosomes by disruption of gene structures. The Bovine-B long interspersed nuclear element (Bov-B LINE) is among the most abundant TE-retrotransposon families in vertebrates due to horizontal transfer (HT) among vertebrate lineages. Recent studies have shown multiple HTs or the presence of diverse Bov-B LINE groups in the snake lineage. It is hypothesized that Bov-B LINEs are highly dynamic and that the diversity reflects multiple HTs in snake lineages. Partial sequences of Bov-B LINE from 23 snake species were characterized. Phylogenetic analysis resolved at least two Bov-B LINE groups that might correspond to henophidian and caenophidian snakes; however, the tree topology differed from that based on functional nuclear and mitochondrial gene sequences. Several Bov-B LINEs of snakes showed greater than 80% similarity to sequences obtained from insects, whereas the two Bov-B LINE groups as well as sequences from the same snake species classified in different Bov-B LINE groups showed sequence similarities of less than 80%. Calculation of estimated divergence time and pairwise divergence between all individual Bov-B LINE copies suggest invasion times ranging from 79.19 to 98.8 million years ago in snakes. Accumulation of elements in a lineage-specific fashion ranged from 9 × 10-6% to 5.63 × 10-2% per genome. The genomic proportion of Bov-B LINEs varied among snake species but was not directly associated with genome size or invasion time. No differentiation in Bov-B LINE copy number between males and females was observed in any of the snake species examined. Incongruence in tree topology between Bov-B LINEs and other snake phylogenies may reflect past HT events. Sequence divergence of Bov-B LINEs between copies suggests that recent multiple HTs occurred within the same evolutionary timeframe in the snake lineage. The proportion of Bov-B LINEs varies among species, reflecting species specificity in TE invasion. The rapid speciation of snakes, coinciding with Bov-B LINE invasion in snake genomes, leads us to better understand the effect of Bov-B LINEs on snake genome evolution.

Acute phase reactions in Daboia siamensis venom and fraction-induced acute kidney injury: the role of oxidative stress and inflammatory pathways in in vivo rabbit and ex vivo rabbit kidney models

Abstract Background: This study examines the direct nephrotoxic effects of Daboia siamensis venom (RVV) and venom fractions in in vivo and isolated perfused kidneys (IPK) to understand the role of inflammation pathways and susceptibility to oxidative stress in venom or fraction-induced acute renal failure. Methods: We administered RVV and its venom fractions (PLA2, MP, LAAO, and PDE) to rabbits in vivo and in the IPK model. We measured oxidative stress biomarkers (SOD, CAT, GSH, and MDA) in kidney tissue, as well as inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-4, IL-5, and IL-10), MDA and GSH levels in plasma and urine. We also calculated fractional excretion (FE) for pro-/anti-inflammatory cytokines and oxidative stress biomarkers, including the ratios of pro-/anti-inflammatory cytokines in urine after envenomation. Results: In both kidney models, significant increases in MDA, SOD, CAT, and GSH levels were observed in kidney tissues, along with elevated concentrations of MDA and GSH in plasma and urine after injecting RVV and venom fractions. Moreover, RVV injections led to progressive increases in FEMDA and decreases in FEGSH. The concentrations of IL-4, IL-5, IL-10, IFN-γ, and TNF-α in plasma increased in vivo, as well as in the urine of the IPK model, but not for IL-1β in both plasma and urine after RVV administrations. Urinary fractional excretion of TNF-α, IL-1β, IFN-γ, IL-4, IL-5, and IL-10 tended to decrease in vivo but showed elevated levels in the IPK model. A single RVV injection in vivo disrupted the balance of urinary cytokines, significantly reducing either the TNF-α/IL-10 ratio or the IFN-γ/IL-10 ratio. Conclusion: RVV induces renal tubular toxicity by increasing oxidative stress production and elevating inflammatory cytokines in urine. During the acute phase of acute kidney injury, the balance of urine cytokines shifts toward anti-inflammatory dominance within the first two hours post-RVV and venom fractions.