Herbal species authentication by melting fingerprint coupled with high resolution melting analysis (MF-HRM).

BACKGROUND: Herbal medicines have recently attracted increasing attention for use as food supplements with health benefits; however, species authentication can be difficult due to incomplete morphological characters. Here, a molecular tool was developed for the identification of species in the National List of Essential Medicinal Plants in Thailand. METHODS: The identification process used DNA fingerprints including start codon targeted (SCoT) and inter simple sequence repeat (ISSR) polymorphisms, coupled with high resolution melting (HRM), to produce melting fingerprint (MF)-HRM. RESULTS: Results indicated that MF-HRM, SCoT-HRM and ISSR-HRM could be used for DNA fingerprints as S34, S36, S9 and S8 of SCoT and UBC873, S25 and UBC841 of ISSR. The melting fingerprints obtained from S34 of SCoT exhibited the best primers for identification of herbal species with 87.5% accuracy and relatively high repeatability. The presence of intraspecific variation in a few species affected the shift of melting fingerprints within species. MF-HRM using S34 showed improved species prediction compared to DNA fingerprints. The concentration of DNA with 10 ng/µl was recommended to perform MF-HRM. MF-HRM enabled species authentication of herbal commercialized products at only 20% resulting from the low quality of DNA isolated, while admixture of multiple product species interfered with the MF process. CONCLUSION: Findings suggested that MF-HRM showed promise as a molecular tool for the authentication of species in commercial herbal products with high specificity, moderate repeatability and rapidity without prior sequence information. This information will greatly improve quality control and traceability during the manufacturing process.

Sparking Nano-Metals on a Surface of Polyethylene Terephthalate and Its Application: Anti-Coronavirus and Anti-Fogging Properties.

The nano-metal-treated PET films with anti-virus and anti-fogging ability were developed using sparking nano-metal particles of Ag, Zn, and Ti wires on polyethylene terephthalate (PET) films. Ag nanoparticles were detected on the PET surface, while a continuous aggregate morphology was observed with Zn and Ti sparking. The color of the Ag-PET films changed to brown with increasing repeat sparking times, but not with the Zn-PET and Ti-PET films. The water contact angle of the nano-metal-treated PET films decreased with increasing repeat sparking times. The RT-PCR anti-virus test confirmed the high anti-virus efficiency of the nano-metal-treated PET films due to the fine particle distribution, high polarity, and binding of the nano-metal ions to the coronavirus, which was destroyed by heat after UV irradiation. A highly transparent, anti-fogging, and anti-virus face shield was prepared using the Zn-PET film. Sparking was an effective technique to prepare the alternative anti-virus and anti-fogging films for medical biomaterial applications because of their low cost, convenience, and fast processing.

Proinflammatory cytokines and lipopolysaccharides up regulate MMP-3 and MMP-13 production in Asian elephant (Elephas maximus) chondrocytes: attenuation by anti-arthritic agents.

BACKGROUND: Osteoarthritis (OA), the most common form of arthritic disease, results from destruction of joint cartilage and underlying bone. It affects animals, including Asian elephants (Elephas maximus) in captivity, leading to joint pain and lameness. However, publications regarding OA pathogenesis in this animal are still limited. Therefore, this study aimed to investigate the effect of proinflammatory cytokines, including interleukin-1 beta (IL-1ß), IL-17A, tumor necrosis factor-alpha (TNF-α), and oncostatin M (OSM), known mediators of OA pathogenesis, and lipopolysaccharides on the expression of cartilaginous degrading enzymes, matrix metalloproteinase (MMP)-3 and MMP-13, in elephant articular chondrocytes (ELACs) cultures. Anti-arthritic drugs and the active compounds of herbal plants were tested for their potential attenuation against overproduction of these enzymes. RESULTS: Among the used cytokines, OSM showed the highest activation of MMP3 and MMP13 expression, especially when combined with IL-1ß. The combination of IL-1ß and OSM was found to activate phosphorylation of the mitogen-activated protein kinase (MAPK) pathway in ELACs. Lipopolysaccharides or cytokine-induced expressions were suppressed by pharmacologic agents used to treat OA, including dexamethasone, indomethacin, etoricoxib, and diacerein, and by three natural compounds, sesamin, andrographolide, and vanillylacetone. CONCLUSIONS: Our results revealed the cellular mechanisms underlying OA in elephant chondrocytes, which is triggered by proinflammatory cytokines or lipopolysaccharides and suppressed by common pharmacological or natural medications used to treat human OA. These results provide a more basic understanding of the pathogenesis of elephant OA, which could be useful for adequate medical treatment of OA in this animal.

Development of 13 microsatellite markers for Castanopsis tribuloides (Fagaceae) using next-generation sequencing.

Catanopsis tribuloides is a climax tree species commonly distributed in evergreen forests and has been used to restore degraded areas in northern Thailand. To aid in study of genetic diversity of the species, microsatellite markers, which are specific to C. tribuloides, were developed using whole genome sequencing by next-generation sequencing technology. The primers for microsatellite were developed and screened for optimal annealing temperature by PCR assay. The loci primers specific with C. tribuloides, 13 polymorphic microsatellite primers were successfully developed. The results from genetic information analyzing showed the number of alleles presented were between 2 and 24. Accordingly, the expected and observed heterozygosity obtained were between 0.298 and 0.920 and 0.364 to 1.000, respectively. Null allele frequency was presented 0.000-0.199. Genetic information was generated 10 loci primers significantly deviated from Hardy-Weinberg Equilibrium. All 13 primer pairs of loci were not significant with linkage disequilibrium. A set of microsatellite markers in this study could be applied to gene flow, genetic structure and population genetic studies in the future.

In Vitro Chondroprotective Potential of Extracts Obtained from Various Phyllantus Species.

Phyllanthus amarus has been proven to exhibit chondroprotection. Regarding the morphological similarities among Phyllanthus species, we were attracted to evaluate the chondroprotective potential of Phyllanthus species including P. amarus obtained from Chiang Mai and Phuket, Phyllanthus urinaria L., Phyllanthus urinaria subsp. chamaepeuce, Phyllanthus debilis, and Phyllanthus airy-shawii using interleukin-1ß-induced degradation of cartilage explants. The ethanolic extracts of the plants were evaluated for major lignans, phyllanthin, and hypophyllanthin by HPLC and further measurements of the total contents of flavonoids and phenolic compounds along with the assays for antioxidant and anti-collagenase activities. The interleukin-1ß-induced cartilage explant degradation was performed with/without the extracts at concentrations of 50-250 µg/mL. After 4-14 days of incubation, the medium was assayed for the level of sulfated glycosaminoglycans while the explants were measured for the remaining content of uronic acid. Proteoglycan intensity in the explants was determined by safranin O staining. Diacerein, the antiarthritic agent, was used as the positive control. Although the two major lignans were found in P. amarus from Chiang Mai, P. amarus from Phuket, and P. urinaria L. extracts, similar chondroprotective activities were observed in all Phyllanthus extracts. Total phenolic content and total flavonoid content of the extracts showed a correlation with antioxidation, whereas the total phenolic content correlated with anti-collagenase activity. Among the six extracts, P. airy-shawii showed the greatest antioxidant and collagenase inhibitory activities. The results revealed that chondroprotective activities of all of the extracts of Phyllanthus species might result from an additive or synergistic influence of some constituents of these plants, which could be considered for antiarthritic purposes.

Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1ß-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1ß, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1ß-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.

Effects of hyaluronic acid combined with anti-inflammatory drugs compared with hyaluronic acid alone, in clinical trials and experiments in osteoarthritis: a systematic review and meta-analysis.

BACKGROUND: The objectives are to compare the efficacy of intra-articular hyaluronic acid (IA-HA) alone and in combination with anti-inflammatory drugs (IA-HA + AI), corticosteroids (CS) or non-steroidal anti-inflammatory drugs (NSAIDs) in clinical trials and in vivo and in vitro studies of osteoarthritis (OA). METHODS: Data in the BIOSIS, CINAHL, Cochrane Library, EMBASE and Medline databases were collected and analyzed. Random effects models were used to compute the effect size (ES) of the mean difference in pain reduction scores from baseline and the relative risk (RR) of adverse events. The ES of histological scores in vivo and cartilage metabolism in vitro were also calculated. We conducted sensitivity analysis of blinding and intention-to-treat (ITT), compared IA-HA combined with CS vs. IA-HA alone in trials, and compared the effects of HA + AI vs. AI alone in vitro, including anabolic and catabolic gene expression. RESULTS: Thirteen out of 382 papers were included for data analysis. In clinical trials, the ES of pain reduction scores within the 1st month was -4.24 (-6.19, -2.29); 2nd-12th month, -1.39 (-1.95, -0.82); and within one year, -1.63 (-2.19, -1.08), favoring IA-HA + AI (P < 0.001). The ES of RR was 1.08 (0.59, 1.98), and histological scores was 1.38 (-0.55, 3.31). The ES of anabolic gene expression was 1.22 (0.18, 2.25), favoring HA alone (P < 0.05); catabolic gene expression was 0.74 (-0.44, 1.53), favoring HA alone; and glycosaminoglycans remaining was -2.45 (-5.94, 1.03). CONCLUSIONS: IA-HA + AI had greater efficacy for pain relief than IA-HA alone within a one-year period. However, HA + AI down-regulated the ACAN gene when compared with HA alone in vitro.

Effects of corticosteroids and their combinations with hyaluronanon on the biochemical properties of porcine cartilage explants.

BACKGROUND: Intra-articular injection of corticosteroids is used to treat the inflammatory pain of arthritis and osteoarthritis (OA), but our previous study found a deleterious effect of these steroids on chondrocyte cells. Hyaluronic acid (HA) injection has been suggested as a means to counteract negative side effects through replenishment of synovial fluid that can decrease pain in affected joints. To better understand the effects of corticosteroids on these processes, dexamethasone (Dex) and prednisolone (Pred) were administered to porcine cartilage explants at several concentrations with and without HA. We examined corticoid effects by determining sulfate-glycosaminoglycan (s-GAG) and uronic acid (UA) content of the explant media, and safranin-O staining of the cells. Analysis of lactate dehydrogenase (LDH) activity was conducted to assess cell cytotoxicity. RESULTS: Dex treatment significantly reduced cellular cytotoxicity compared to the other treatment groups, especially with regards to the release of s-GAG, and protects against superficial proteoglycan damage. However, there was no difference between Pred and Dex, with and without HA, in the UA content remaining in porcine cartilage explants. CONCLUSIONS: The data suggest that combinations of Dex and Pred with HA did not have a significant effect on protection or enhancement of the articular cartilage matrix under the current conditions.

Monascus purpureus-fermented Thai glutinous rice reduces blood and hepatic cholesterol and hepatic steatosis concentrations in diet-induced hypercholesterolemic rats.

BACKGROUND: Red yeast rice (RYR) is a fermented product used as a food supplement to promote blood circulation and lower blood cholesterol levels in eastern Asia. Interestingly, monacolin K is the most active compound in RYR that proved to inhibit HMG-CoA reductase in the cholesterol biosynthesis pathway. METHODS: The hypocholesterolemic effects of oral administration of Thai RYR, produced by fermentation of Thai glutinous rice (Oryza sativa L. var. Niaw San-pah-tawng) with Monascus purpureus CMU 002U, were determined in normal and hypercholesterolemic rats. The rats were divided into six groups, and fed two different kinds of diet. Groups I-II, normal rats fed with a normal diet (SP-diet), were treated with distilled water (SP-control) and 2.0 g/kg/day of RYR extract (SP-2 g). In Groups III-VI, the rats were rendered hypercholesterolemic by feeding them a high fat and cholesterol diet (HFC-diet), and were treated with distilled water (HFC-control), 1.0 g/kg/day (HFC-1 g), 2.0 g/kg/day (HFC-2 g) of RYR extract, and 5.0 mg/kg/day of rosuvastatin (HFC-rosuvastatin) for 30 days, respectively. RESULTS: The RYR extract significantly decreased the concentrations of serum total cholesterol and low density lipoprotein cholesterol (LDL-C), atherosclerotic index, LDL-C/HDL-C ratio and hepatic cholesterol levels in both HFC-1 g and HFC-2 g groups (p < 0.05) as compared with the HFC-control group, and with no significant change in high density lipoprotein cholesterol (HDL-C) concentrations among all six groups. The reduction of serum TC and LDL-C also paralleled the observed changes in mRNA expressions of the genes involved in cholesterol biosynthesis and homeostasis in the liver. The hypercholesterolemic rats treated with RYR extract were significantly higher in LDLR and HMGR expression, but lower in CYP7A1 expression when compared to the untreated hypercholesterolemic rats (HFC-control) (p < 0.05). The hepatic injuries in hypercholesterolemic rats were also obviously alleviated by RYR extract. CONCLUSIONS: The extract of Thai RYR possessed potent hypocholesterolemic and anti-atherogenic activities in diet-induced hypercholesterolemic rats. The possible mechanism involving cholesterol-lowering potential of the extract might contribute to its ability to increase LDL-C endocytosis in hepatocyte and to competitively inhibit HMG-CoA reductase, a key enzyme for cholesterol biosynthesis in liver.

A modified colorimetric method of gelatinolytic assay using bacterial collagenase type II as a model.

Coomassie brilliant blue (CBB) heated by microwave was used as a staining dye for measuring gelatinolytic activity. The quantity of gelatin remaining after incubation with bacterial collagenase was determined using the heated CBB, resulting in visible blue pellets. Dimethyl sulfoxide was added to dissolve the dye and measurement of the absorbance at 600 nm was done to detect the level of gelatin (up to 10 µg), with the limit of detection for the amount of collagenase at 50 ng. This approach is rapid, simple, and economic for the purpose of screening for pharmaceutical agents that possess inhibitory activity on collagenase.

A new species of the Cyrtodactylusbrevipalmatus group (Squamata, Gekkonidae) from Tak Province, northwestern Thailand.

An integrative taxonomic analysis was used to delimit and diagnose a new species of the Cyrtodactylusbrevipalmatus group from Tak Province in western Thailand. Although Bayesian phylogenetic analyses place C.denticulatussp. nov. within the brevipalmatus group, the new species is neither nested within nor is it the sister species of any other species in the brevipalmatus group. Furthermore, based on the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs, it bears an uncorrected pairwise sequence divergence of 7.87-21.94% from all other species in the brevipalmatus group. Cyrtodactylusdenticulatussp. nov. is differetiated from all other species in the brevipalmatus group by having a number of unique charateristics such as denticulate ventrolateral body folds and ventrolateral subcaudal ridges, characters not seen in any other species of the group (n = 51 individuals). Additionally, based on a multiple factor anlaysis, C.denticulatussp. nov. does not overlap with any other species in multivariate space. The discovery of C.denticulatussp. nov. underscores the unrealized diversity of upland ecosystems across Thailand and the urgent need for increased exploration and conservation of these unique imperiled montane refugia, especially in this era of climate change.

A new species of the Cyrtodactylusbrevipalmatus group (Squamata, Gekkonidae) from the uplands of western Thailand.

An integrative systematic analysis recovered a new species of the Cyrtodactylusbrevipalmatus group from the uplands of Thong Pha Phum National Park, Kanchanaburi Province in western Thailand. Cyrtodactylusthongphaphumensissp. nov. is deeply embedded within the brevipalmatus group, bearing an uncorrected pairwise sequence divergence of 7.6-22.3% from all other species based on a 1,386 base pair segment of the mitochondrial NADH dehydrogenase subunit 2 gene (ND2) and adjacent tRNAs. It is diagnosable from all other species in the brevipalmatus group by statistically significant mean differences in meristic and normalized morphometric characters as well as differences in categorical morphology. A multiple factor analysis recovered its unique and non-overlapping placement in morphospace as statistically significantly different from that of all other species in the brevipalmatus group. The description of this new species contributes to a growing body of literature underscoring the high degree of herpetological diversity and endemism across the sky-island archipelagos of upland montane tropical forest habitats in Thailand, which like all other upland tropical landscapes, are becoming some of the most imperiled ecosystems on the planet.

RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand.

Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37 °C for 20 min, followed by CRISPR-Cas12a digestion at 37 °C for one hour. A combination of RPA and the cas12a detection method did not react with other pathogens and demonstrated strong sensitivity, detecting as low as 100 copies of both E. canis and A. platys. This simultaneous detection method was significantly more sensitive than conventional PCR. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple and appropriate detection of rickettsial agents in canine blood at the point-of-care for diagnostics, disease prevention and surveillance.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Microsatellite Polymorphism and the Population Structure of Dugongs (Dugong dugon) in Thailand.

The dugong (Dugong dugon) is an endangered species of marine mammals, so knowledge of genetic diversity of these populations is important for conservation planning within different habitats. In this study, six microsatellite markers were used to assess the genetic diversity and population structure of 77 dugongs from skin samples of stranded animals collected from 1994-2019 (69 from Andaman Sea and 8 from the Gulf of Thailand). Our results found that dugongs in the Andaman Sea had higher genetic variation than those in the Gulf of Thailand. Populations in Trang, Satun, and some areas of Krabi had highest diversity compared to other regions of Thailand. Bayesian genetic clustering analysis revealed that dugongs in Thailand consist of five genetic groups. Moreover, dugongs in the middle and lower Andaman Sea presented the greatest gene flow compared to other regions. However, based on calculation of inbreeding coefficients (Fis value = 0.239), dugong populations in the Sea of Thailand are experiencing some levels of inbreeding, and so may warrant special protections. These results provide important information for understanding the genetic status of dugongs that can lead to improved management and conservation of this endangered species.

Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/µl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.

Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.

Mammalian species identification using ISSR-HRM technique.

Wildlife trading and the illegal hunting of wildlife are contributing factors to the biodiversity crisis that is presently unfolding across the world. The inability to control the trade of animal body parts or available biological materials is a major challenge for those who investigate wildlife crime. The effective management of this illegal trade is an important facet of wildlife forensic sciences and can be a key factor in the enforcement of effective legislation surrounding the illegal trade of protected and endangered species. However, the science of wildlife forensics is limited by the absence of a comprehensive database for wildlife investigations. Inter-simple sequence repeat markers (ISSR) coupled with high resolution melting analysis (HRM) have been effectively used for species identification of 38 mammalian species. Six primers of the ISSR markers were chosen for species identification analysis. From six ISSR primers resulting in a range of accuracy of 33.3%-100% and 100% in terms of precision in every primer. Furthermore, 161 mammalian samples were 100% distinguished to the correct species using these six ISSR primers. ISSR-HRM analysis was successfully employed in determining mammal identification among varying mammalian species, and thus could serve as an effective alternative tool or technique in the species identification process. This option would offer researchers a heightened level of convenience in terms of its performance and the ease with which researchers or field practice veterinarians would be able to interpret results in effectively identifying animal parts at wildlife investigation crime scenes.

Phylogenetic analyses of distantly related clades of bent-toed geckos (genus Cyrtodactylus) reveal an unprecedented amount of cryptic diversity in northern and western Thailand.

Cyrtodactylus species are the most diverse of the geckos and are widely distributed in Southeast Asia, including Thailand. However, their patterns of distribution, especially in northern and western parts of Thailand, remain unknown because few Cyrtodactylus species in these regions have been described. Thus, a data set of mitochondrial NADH dehydrogenase 2 (ND2) gene and flanking tRNAs from Cyrtodactylus found in northern and western Thailand, including contiguous areas, was assembled to elucidate phylogenetic relationships and identify the distribution patterns of these geckos. The results showed four well-supported clades, a northwestern clade (A), a northern clade (B), a western clade (C), and a special clade characterized by specific morphological features (D). Clades A-C were grouped with strong support by the geography of their localities from northern Thailand (Mae Hong Son and Chiang Mai Provinces) along the Tenasserim mountain ranges to Phang-Nga Province, Thailand. Clade D is a distinct clade of Cyrtodactylus species characterized by a tuberculate and prehensile tail and distributed widely in mainland Southeast Asia. Overall, the results suggest a pattern of geographic separation and distribution of Cyrtodactylus in northern and western Thailand. Additionally, this study provides evidence of a hidden biodiversity of Cyrtodactylus in these regions.