Species discrimination from hair using ATR-FTIR spectroscopy: Application in wildlife forensics.

Hair is a commonly encountered trace evidence in wildlife crimes involving mammals and can be used for species identification which is essential for subsequent judicial proceedings. This proof of concept study aims, to distinguish the black guard hair of three wild cat species belonging to the genus Panthera i.e. Royal Bengal Tiger (Panthera tigris tigris), Indian Leopard (Panthera pardus fusca), and Snow Leopard (Panthera uncia) using a rapid and non-destructive ATR-FTIR spectroscopic technique in combination with chemometrics. A training dataset including 72 black guard hair samples of three species (24 samples from each species) was used to construct chemometric models. A PLS2-DA model successfully classified these three species into distinct classes with R-Square values of 0.9985 (calibration) and 0.8989 (validation). VIP score was also computed, and a new PLS2DA-V model was constructed using variables with a VIP score ≥ 1. External validation was performed using a validation dataset including 18 black guard hair samples (6 samples per species) to validate the constructed PLS2-DA model. It was observed that PLS2-DA model provides greater accuracy and precision compared to the PLS2DA-V model during cross-validation and external validation. The developed PLS2-DA model was also successful in differentiating human and non-human hair with R-Square values of 0.99 and 0.91 for calibration and validation, respectively. Apart from this, a blind test was also carried out using 10 unknown hair samples which were correctly classified into their respective classes providing 100 % accuracy. This study highlights the advantages of ATR-FTIR spectroscopy associated with PLS-DA for differentiation and identification of the Royal Bengal Tiger, Indian Leopard, and Snow Leopard hairs in a rapid, accurate, eco-friendly, and non-destructive way.

Preliminary study on cranial measurements and sexual dimorphism in skull bones of gaur (Bos gaurus gaurus, Smith 1827).

Cranial measurements have been widely used in various studies in wildlife sciences, ranging from understanding predator ecology to wildlife forensics. However, detailed description of morphometry and sexual dimorphism of the skull of gaur Bos gaurus gaurus is lacking. The present study was undertaken to determine the sexual dimorphism based on the cranial measurements of gaur. A total of 12 individual gaur skulls of male (n = 6) and female (n = 6) were studied in the field from the naturally deceased animals between January 2018 and December 2021 in different ranges of Bandhavgarh tiger reserve (BTR), Madhya Pradesh, India. The skull measurements were analysed using univariate and multivariate statistics to determine whether cranial dimensions could be used to differentiate male and female skulls reliably. A total of 43 morphometrical parameters grouped into nine indices were calculated. Select morphometrical parameters viz PL, GFL, AKI, LBB, LFB, GBEE, GBAN, BPOP and GTCH were significantly different (p < 0.05) between sexes, whereas GBAN were significantly higher in female skulls. The measurements demonstrated that the skull of the gaur was dolichocephalic as the profile length and the otion to otion breath in both male and female were <75% of the length. Overall, 28 linear measurements of both the sexes were statistically significant (p < 0.05; <0.01). The calculated indices revealed that the foramen magnum index in the female gaur were significantly higher. In calculated cranial indices the facial index (a) was higher in female and facial index (b) were higher in males. The two important parameters, facial breadth in facial index (a) and the greatest breadth in facial index (b) were positively correlated, though facial index (a) was statistically not significant between the sexes. The greater inner length of the foramen magnum in female skull resulted in foramen being oval whereas it was circular in males. These parameters were decisive for sexual dimorphism, skull comparison and craniological studies. This study ascertained that the frontal index and skull index had no significant influence and were not good indices for discriminating skulls between male and female. Based on the Principal Component Analysis, it was found that skull of male and female gaurs exhibits differences in cranial morphology viz. cranial profile length or total length (PL) and the least inner height of the temporal groove (LIHT). The findings of the present study provide baseline information on various craniometrical measurements of skull of gaur, indices and parameters for sex identification that can be effectively used in understanding sex biased predation ecology, provide base line information to describe variation across its geographic range, and in identifying skulls recovered in wildlife offence cases.

A new class of teraryl-based AIEgen for highly selective imaging of intracellular lipid droplets and its detection in advanced-stage human cervical cancer tissues.

Lipid droplets (LDs) have drawn much attention in recent years. They serve as the energy reservoir of cells and also play an important role in numerous physiological processes. Furthermore, LDs are found to be associated with several pathological conditions, including cancer and diabetes mellitus. Herein, we report a new class of teraryl-based donor-acceptor-appended aggregation-induced emission luminogen (AIEgen), 6a, for selective staining of intracellular LDs in in vitro live 3T3-L1 preadipocytes and the HeLa cancer cell line. In addition, AIEgen 6a was found to be capable of staining and quantifying the LD accumulation in the tissue sections of advanced-stage human cervical cancer patients. Unlike commercial LD staining dyes Nile Red, BODIPY and LipidTOX, AIEgen 6a showed a high Stokes shift (195 nm), a good fluorescence lifetime decay of 12.7 ns, and LD staining persisting for nearly two weeks.

Picomolar Detection of Lead Ions (Pb2+) by Functionally Modified Fluorescent Carbon Quantum Dots from Watermelon Juice and Their Imaging in Cancer Cells.

Water contamination due to the presence of lead is one of the leading causes of environmental and health hazards because of poor soil and groundwater waste management. Herein we report the synthesis of functionally modified luminescent carbon quantum dots (CQDs) obtained from watermelon juice as potential nanomaterials for the detection of toxic Pb2+ ions in polluted water and cancer cells. By introducing surface passivating ligands such as ethanolamine (EA) and ethylenediamine (ED) in watermelon juice, watermelon-ethanolamine (WMEA)-CQDs and watermelon-ethylenediamine (WMED)-CQDs exhibited a remarkable ~10-fold and ~6-fold increase in fluorescence intensity with respect to non-doped WM-CQDs. The relative fluorescence quantum yields of WMEA-CQDs and WMED-CQDs were found to be 8% and 7%, respectively, in an aqueous medium. Among various functionally-modified CQDs, only WMED-CQDs showed high selectivity towards Pb2+ ions with a remarkably good limit of detection (LoD) of 190 pM, which is less than that of the permissible limit (72 nM) in drinking water. The functionally altered WMED-CQDs detected Pb2+ metal ions in polluted water and in a human cervical cancer cell line (HeLa), thus advocating new vistas for eco-friendly nanomaterials for their use as diagnostic tools in the environment and biomedical research areas.

Avian Haemosporidian (Plasmodium and Haemoproteus) Status in Two Bird Groups (Old-World Flycatchers and Thrushes) of India and Their Phylogenetic Relationships with Other Lineages of the World.

PURPOSE: Avian haemosporidian may affect the host from body damage to the extinction of a population. Knowledge of their status may help in future avifauna conservation plans. Hence, their status in two bird groups of India and their phylogenetic relationships with other known lineages of the world were examined. METHODS: Cytochrome b gene sequences (479 bp) generated from India and available at MalAvi database were used to study the avian haemosporidian prevalence and phylogenetic analysis of lineages at local and world levels. RESULTS: One common (COLL2) and only once in the study (CYOPOL01, CHD01, CYORUB01, EUMTHA01, GEOCIT01) haemosporidian lineages were discovered. 5.88% prevalence of haemosporidian infection was found in 102 samples belonging to 6 host species. Haemoproteus prevalence was 4.90% across five host species (Phylloscopus trochiloides, Cyornis poliogenys, C. hainanus dialilaemus, C. rubeculoides, Eumiyas thalassinus) and Plasmodium prevalence was 0.98% in Geokichla citrina. Spatial phylogeny at the global level showed that COLL2 lineage, found in C. poliogenys in India, was genetically identical to H. pallidus lineages (COLL2) in parts of Africa, Europe, North America, Malaysia, and the Philippines. The Plasmodium lineage (GEOCIT01) was related to PADOM16 in Egypt, but the sequences were only 93.89% alike. CONCLUSIONS: Four new lineages of Haemoproteus and one of Plasmodium were reported. COLL2 similarity with other H. pallidus lineages may suggest their hosts as possible infection sources.

Unraveling the mystery of confiscated "jackal horns" in India using wildlife forensic tools.

Internationally, illegal wildlife trade involves highly prized and charismatic species and their derivatives. At the same time, common or less known species and their parts are also encountered but receive less attention than charismatic species. Given the increasing demand for wildlife products in many parts of the world, profit, and short supply, many fake articles derived from domestic or wild animals are frequently encountered in the wildlife trade. Jackal horn (locally known as "Siyar or Gidar singhi") is one such fake item widely used in sorcery and other occult practices available through offline and online trading platforms within India. We used a combination of morphological, microscopic hair, and molecular approaches (Cyt b and 16 s rRNA genes) to reveal the true identity of confiscated "jackal horns" (n = 342). Detailed morphological study of the jackal horns showed that it varied in size, shape, color of hair, attachment material, and filling material. The microscopic hair and molecular approaches revealed that all the items sold as jackal horns were fake and made up of protected wild species and domestic animals. Our results confirm the use of the biological samples from few wild species protected under the Wild Life (Protection) Act, 1972, of India. Therefore, the law enforcement agencies are cautioned to get forensic opinions while dealing with such counterfeit items.

Design, synthesis, in vitro and in vivo biological evaluation of pyranone-piperazine analogs as potent antileishmanial agents.

The current therapeutic regimen for visceral leishmaniasis is inadequate and unsatisfactory due to toxic side effects, high cost and emergence of drug resistance. Alternative, safe and affordable antileishmanials are, therefore, urgently needed and toward these we synthesized a series of arylpiperazine substituted pyranone derivatives and screened them against both in vitro and in vivo model of visceral leishmaniasis. Among 22 synthesized compounds, 5a and 5g showed better activity against intracellular amastigotes with an IC50 of 11.07 µM and 15.3 µM, respectively. In the in vivo, 5a significantly reduced hepatic and splenic amastigotes burden in Balb/c mice model of visceral leishmaniasis. On a mechanistic node, we observed that 5a induced direct Leishmania killing via mitochondrial dysfunction like cytochrome c release and loss of membrane potential. Taken together, our results suggest that 5a is a promising lead for further development of antileishmanial drugs.

Identification of selected wild felids using hair morphology and forensically informative nucleotide sequencing (FINS): Wildlife forensics prospective.

We describe the identification of the leopard cat (Prionailurus bengalensis), jungle cat (Felis chaus) fishing cat (Prionailurus viverrinusis), wild cat (Felis silvestris) and caracal (Caracal caracal) using combined approach of morphological and molecular based analysis. Three mitochondrial genes [12S rRNA, 16S rRNA and cytochrome b (Cyt b)] and hair characteristics (cuticle and medulla patterns) were analysed and variation was observed among few selected wild felids. We did not find conclusive morphological difference among these examined felids. We observed maximum variation in Cyt b in comparison to12S rRNA and 16S rRNA genes. Therefore, despite poor hair morphological difference, forensically informative nucleotide sequencing (FINS) exhibited unambiguous variation among the examined felids. We suggest the use of FINS for differentiating biological samples of closely related wild felids to avoid any false identification of species in illicit trade. Furthermore, the data generated from the present work would help in strengthening the DNA database of Indian small cats.

Sentinel Lymph Node Biopsy in Colon Cancer: an Institutional Experience.

Lymph node staging is a major prognostic factor in colorectal cancer and remains to be the most important criterion for selecting patients for adjuvant therapy. The standard approach for lymph node evaluation is based on manual dissection and histological evaluation of HE-stained slides. For stage III disease (node positive), adjuvant chemotherapy increases the survival rate, while in node-negative stage II disease, in most cases, the chemotherapy is contraindicated due to increased morbidity without real benefit. Up to 30% of patients with node-negative colon cancer staged by standard pathologic techniques ultimately suffer disease recurrence and tumour-related mortality following potentially curative primary resection. Variations in outcome among patients with node-negative early-stage disease may reflect inadequate nodal resection and inaccuracies of pathologic staging. Hence, an accurate pN stage becomes essential. It is seen that classic pathological exam sometimes fails to identify lymph node micrometastases or isolated tumour cells, which might explain local or distant relapses in stage II patients. Sentinel lymph node study has the potential to detect micrometastases and lead to upstaging the disease which is crucial for planning adjuvant therapy and follow-up in these patients. In our study, we carried out SLNB in 40 clinically stage II patients operated for colon cancer. We used peritumoural injection of dye at the time of surgery to detect SLN(s) and analysed them using both microsectioning and immunohistochemical (IHC) staining. Our results show that SLNB can improve the accuracy of pTNM staging.

Rapid and non-destructive identification of claws using ATR-FTIR spectroscopy-A novel approach in wildlife forensics.

Differentiation and identification of Royal Bengal Tiger (Panthera tigris tigris) and Indian Leopard (Panthera pardus fusca) claws is a challenging task in wildlife forensics, due to similarity in their morphology, anatomy and chemical compositions as both the species are closely related to each other genetically. ATR-FTIR spectroscopy, which offers a non-destructive and safe alternative technique to other conventional methods, has been employed in the present work to differentiate claws of Royal Bengal Tiger and Indian Leopard. An attempt has been made to differentiate 31 reference claw samples from 16 different Royal Bengal Tigers, 15 different Indian Leopards, and 10 fake claws using ATR-FTIR spectroscopy supplemented with PCA, PLS-DA, and LDA. PCA could not distinguish the samples of two closely related species among themselves as well as from the fake claws. On the other hand, PLS-DA and LDA models both yielded highly significant classification rate for differentiation among the samples of Royal Bengal Tiger, Indian Leopard, and their fake counterparts. Further, seven blind claw samples that were pretended to be unknown to the analyst of both the species are also examined and identified correctly to their respective groups. The R-Square value obtained for PLS-DA model to differentiate Royal Bengal Tiger, Indian Leopard, and fake claws is 0.99, which is highly significant for predictive accuracy. This study shows that ATR-FTIR spectroscopy with PLS-DA/LDA has a potential to present a rapid, non-destructive, reliable, and eco-friendly approach for the accurate identification and differentiation of Royal Bengal Tiger and Indian Leopard claws.

Online selling of wildlife part with spurious name: a serious challenge for wildlife crime enforcement.

We examined an online sold product "Hatha Jodi" synonym of "paired arm" for the confirmation of its biological source. It was declared as a plant root. The morphological features of these samples were matched with the "intromittent organs" or "hemi penis" of the monitor lizard. For further confirmation, we used sequencing of a partial fragment of mitochondrial DNA cytochrome b gene. Sequence comparison indicated that these claimed plant products were actually biological samples of a common monitor lizard, Varanus bengalensis. Hence, it exhibited the ongoing illegal trade of the intromittent organ of a prohibited species with a misleading name using low risk and widely adopted modern trading method that imposes a severe challenge for combating against the wildlife crime.

Pioneer identification of fake tiger claws using morphometric and DNA-based analysis in wildlife forensics in India.

The illegal trade in wildlife is a serious threat to the existence of wild animals throughout the world. The short supply and high demand for wildlife articles have caused an influx of many different forms of fake wildlife articles into this trade. The task of identifying the materials used in making such articles poses challenges in wildlife forensics as different approaches are required for species identification. Claws constitute 3.8% of the illegal animal parts (n=2899) received at the Wildlife Institute of India (WII) for species identification. We describe the identification of seized suspected tiger claws (n=18) using a combined approach of morphometric and DNA-based analysis. The differential keratin density, determined using X-ray radiographs, indicated that none of the 18 claws were of any large cat but were fake. We determined three claw measurements, viz. ac (from the external coronary dermo-epidermal interface to the epidermis of the skin fold connecting the palmar flanges of the coronary horn), bc (from the claw tip to the epidermis of the skin fold connecting the palmar flanges of the coronary horn) and the ratio bc/ac, for all the seized (n=18), tiger (n=23) and leopard (n=49) claws. Univariate and multivariate statistical analyses were performed using SPSS. A scatter plot generated using canonical discriminant function analysis revealed that of the 18 seized claws, 14 claws formed a cluster separate from the clusters of the tiger and leopard claws, whereas the remaining four claws were within the leopard cluster. Because a discrepancy was observed between the X-ray images and the measurements of these four claws, one of the claw that clustered with the leopard claws was chosen randomly and DNA analysis carried out using the cyt b (137bp) and 16S rRNA (410bp) genes. A BLAST search and comparison with the reference database at WII indicated that the keratin material of the claw was derived from Bos taurus (cattle). This is a pioneering discovery, and we suggest that a hierarchical combination of techniques be used for identifying claws involved in wildlife offences, i.e. that an X-ray, morphometric and DNA-based analysis be carried out, to ascertain whether the claws are of tigers or leopards. To identify species in the illegal wildlife trade morphometric and genetic reference database should be developed. Morphological features as well as DNA profiles need to be used for better implementation of the Wildlife (Protection) Act, 1972 of India and other laws/treaties in South-east Asia.

Blood compatibility studies of Swarna bhasma (gold bhasma), an Ayurvedic drug.

Swarna bhasma (gold bhasma) preparations are widely utilized as therapeutic agents. However, in vitro biological evaluations of bhasma preparations are needed along with the physicochemical characterization for present day standardization of metallic bhasma preparations to meet the criteria that supports its use. Therefore, an attempt has been made to evaluate the protein adsorption, blood compatibility and complement activation potential of two batches of Swarna bhasma preparation, along with its physicochemical characterization. The particle size, morphology, elemental analysis, and in vitro cytotoxicity were evaluated initially. Red blood cell hemolysis, aggregation studies with blood cells, protein adsorption, complement C3 adsorption, platelet activation and tight junction permeability in Caco-2 cell line were investigated. The Swarna bhasma preparations with a crystallite size of 28-35 nm did not induce any blood cell aggregation or protein adsorption. Activation potential of these preparations towards complement system or platelets was negligible. These particles were also non-cytotoxic. Swarna bhasma particles opened the tight junctions in Caco-2 cell experiments. The results suggest the application of Swarna bhasma preparations as a therapeutic agent in clinical medicine from the biological safety point of view.

Zinc is critically required for pollen function and fertilisation in lentil.

The role of zinc (Zn) in reproduction of lentil (Lens culinaris Medik. cv. DPL 15) and the extent to which the Zn requirement for reproduction can be met through supplementation of Zn at the time of initiation of the reproductive phase have been investigated. Low supply (0.1micromol/L) of Zn reduced the size of anthers, the pollen producing capacity and the size and viability of the pollen grains. Scanning electron microscopy (SEM) of pollen grains of Zn deficient plants showed enhanced thickening of exine and wide and raised muri. In vitro germination of pollen grains was reduced by >50% and growth of pollen tubes was retarded. Unlike Zn sufficient plants, the cuticle around the stigmatic papillae of Zn deficient plants remained intact, preventing the interaction between pollen grains and stigmatic exudates that provides the polarity for the growth of pollen tubes through the stylar tract. Zn deficiency increased the activity of acid phosphatase and peroxidase in extracts of pollen grains. Histochemical localisation on the stigmatic surface and native PAGE of the enzyme extracts of pollen grain and stigma exudates showed enhanced expression of acid phosphatase and peroxidase and suppressed expression of esterase in response to Zn deficiency. Zn deficiency reduced the setting of seeds and also their viability. The effect on seed setting was more marked than on in vitro germination of pollen grains, suggesting that the latter was not the exclusive cause of inhibition of fertility. Possibly, loss of fertility was also caused by impairment in pollen-pistil interaction conducive to pollen tube growth and fertilisation. Impairment in pollen structure and function and seed setting was observed even when plants were deprived of Zn at the time of flowering, but to a lesser extent than in plants maintained with low Zn supply from the beginning. Increasing the Zn supply from deficient to sufficient at the initiation of flowering decreased the severity of Zn deficiency effects on pollen and stigma morphology, pollen fertility and seed yield. In conclusion, structural and functional changes induced in pollen grains and stigma of Zn deficient plants and associated decrease in seed setting of lentil indicate a critical requirement of Zn for pollen function and fertilisation that can be partially met by supplementing Zn at the onset of the reproductive phase.