Reactive nitrogen species-mediated cell proliferation during tail regeneration and retinoic acid as a putative modulator of tissue regeneration in the geckos.

Reactive nitrogen species (RNS), a mediator of nitrosative stress, plays a vital role during wound healing but its role during tissue regeneration is poorly understood. In the present study, the role of RNS was investigated post-tail autotomy and limb amputation in a gecko species, Hemidactylus murrayi Gleadow, 1887. Tail autotomy led to an increased expression of iNOS and nitrosative stress leading to protein S-nitrosylation that probably restricted the acute inflammatory response caused by wounding. Increased nitrosative stress was also associated with proliferation of the wound epithelium and the tail blastema. Nitric oxide synthase inhibitor (L-NAME) caused retarded growth and structural abnormalities in the regenerating tail while peroxynitrite inhibitor (FeTmPyp) arrested tail regeneration. Spermine NONOate and retinoic acid, used as NO donors generated small outgrowths post-amputation of limbs with an increased number of proliferating cells and s-nitrosylation indicating the role of nitric oxide signalling in cell proliferation during regeneration. Additionally, retinoic acid treatment caused regeneration of nerve, muscle and adipose tissue in the regenerated limb structure 105 days post-amputation suggesting it to be a putative modulator of tissue regeneration in the non-regenerating limbs.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Protein S-nitrosylation: Nitric oxide signalling during anuran tail regression.

Tail regression is a remarkable process where a complex organ like the tail is completely resorbed by cell death during anuran metamorphosis. Nitric oxide is a signalling molecule involved in various physiological processes and along with reactive nitrogen species induces apoptosis. The present study describes the contribution of nitric oxide and reactive nitrogen species (nitrosative stress) during tail regression in the tadpoles of Indian tree frog, Polypedates maculatus. Spectrophotometric estimation revealed significantly higher levels of nitrite, nitrate and peroxynitrite in the regressing tails of the late climactic stages as compared to the early climactic stages and pre-regressing tails. S-nitrosylated proteins were detected in the apoptotic cells of epidermis and muscle, denervated and fragmented myofibres, outer notochordal sheath of the degenerating notochord, endothelium of blood vessels, blood cells and spinal cord of the regressing tail of the late climactic stages using fluorescent detection methods. Thus, a higher level of nitrosative stress in the late climactic stages is suggested to cause S-nitrosylation of proteins and subsequent apoptosis in the tail tissues. Macrophages were found engulfing the apoptotic cells and cell debris at the distal end of the regressing tail. Interestingly, macrophages were always found to be associated with melanocytes suggesting a close association for clearing cell debris by phagocytosis.

Physiological assessment of fish health in mineral-rich areas of Ganjam, Odisha, India, and chronic toxicity of zirconium oxychloride on the fishes of Channa punctata.

The high concentration of metal toxicants in aquatic ecosystems has a detrimental impact on fish health that ultimately jeopardizes human health. Such threats mostly arise in mineral-rich areas where an increase in metal concentrations occurs in aquatic bodies due to anthropogenic activities like mining. The present study assessed the health of food fish Channa punctata from the mineral-rich areas of Ganjam, Odisha, India, mined for heavy and transition metal ores like ilmenite, zircon, monazite. The fishes collected from these areas showed histopathological anomalies in vital organs like the liver, kidney, gills, stomach and intestine while cytological analysis revealed vacuolated cytoplasm and micronuclei. Biochemical analysis showed a significantly lower lipid concentration in muscle (i.e., 0.177 ± 0.177 mg/gm) and liver (i.e., 0.169 ± 0.002 mg/gm) as compared to non-exposed fishes from adjoining non-mineral rich areas having a mean protein concentration of 87.48 ± 8.16 and 77.75 ± 0.892 mg/gm tissue in the muscle and liver, respectively, and a mean lipid concentration of 0.29 ± 0.009 mg/gm muscle and 0.34 ± 0.009 mg/gm liver. Chronic exposure to sublethal concentrations of zirconium oxychloride, a salt of zirconium, resulted in a significant decline in the concentration of protein, ranging from 57.5 ± 0.929 - 63.88 ± 1.95 mg/gm in muscle and 45.35 ± 2.332 - 51.98 ± 1.036 mg/gm in the liver. The lipid concentrations in muscle (0.03 ± 0.009 - 0.17 ± 0.009 mg/gm) and liver (0.06 ± 0.012 - 0.19 ± 0.007 mg/gm) were also significantly lower than the non-exposed fishes. Marked degenerative changes were identified in the histological sections of the gill, intestine, stomach, liver and kidney of zirconium oxychloride-treated fishes along with various nuclear deformities and micronucleus.

Description of three new species of the genus Mata Distant, 1906 (Hemiptera: Cicadidae: Cicadinae: Oncotympanini) with notes on their natural history from Indian state of Meghalaya, India.

Three new species of the Asian genus Mata Distant, 1906 (Hemiptera: Cicadidae) viz. Mata lenonia sp.nov.; Mata ruffordii sp.nov. and Mata meghalayana sp.nov. are described from Indian state of Meghalaya. Keys and taxonomic descriptions of these species are provided with detailed accounts of their natural history and acoustics.

A novel, diminutive Hemiphyllodactylus Bleeker, 1860 (Squamata: Gekkonidae) from a sacred grove in Odisha, eastern India.

We describe a distinct new species of Hemiphyllodactylus from a sacred grove near Humma in Ganjam District of Odisha state, eastern India. Hemiphyllodactylus minimus sp. nov. is the smallest member of the genus and can be diagnosed from all peninsular Indian congeners by its small body size (SVL ≤32.2 mm, n=11), having 15-18 dorsal scales and eight or nine ventral scales at mid-body contained within one longitudinal eye diameter, nine or ten precloacal pores separated by 4-6 poreless scales from a series of six or seven femoral pores on each thigh in males, lamellar formula of manus 2222 and of pes 2332 2333, as well as subtle colour pattern differences. Additionally, the new species is 16.0-22.7 % divergent from all other peninsular Indian Hemiphyllodactylus in uncorrected ND2 sequence data. The new species is the first member of the genus described from Odisha state and also the first Hemiphyllodactylus known from < 100 m asl. The discovery of Hemiphyllodactylus minimus sp. nov. from a low elevation coastal locality in the Eastern Ghats open ups the possibilities that the genus could be more widespread than currently understood and potentially harbour many more distinct undescribed lineages. Therefore, dedicated fine-scale sampling efforts are needed to uncover the true diversity and distribution of Hemiphyllodactylus in the region.

Immunohistochemical localization of cathepsin D and a possible role of melanocytes during tail resorption in tadpoles of a tropical toad.

Programmed cell death during anuran tail resorption is primarily brought about by apoptosis. Cathepsin D, a lysosomal aspartyl protease, is involved in the death of tail tissues. Thus, anuran tail resorption presents an ideal model to study cathepsin-mediated cell death during vertebrate development. Present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D in the tail tissues of the common Asian toad, Duttaphrynus melanostictus. Cathepsin D was involved in programmed cell death in epidermis, muscle, spinal cord, and blood cells in the resorbing tail. Interestingly, it was also involved in the pre-resorbing tail before visible tail resorption which indicates initiation of cell death even before actually the tail resorbs. Melanocytes were found to be one of the causative agents in degrading tail tissues and were associated with the degradation of muscle, epidermis and spinal cord of the resorbing tail.

Involvement of cathepsin D during tail regression in tadpoles of the common Indian tree frog, Polypedates maculatus (Anura: Rhacophoridae).

Cathepsin D, an aspartyl protease, plays a key role in the metabolic degradation of intracellular proteins in an acidic milieu of lysosomes. Proteolysis plays an essential role in anuran tail regression and a wide variety of thyroid hormone induced proteolytic enzymes have been reported to be involved in the regressing tail. The present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D during degradation of various tail tissues in the tadpoles of Polypedates maculatus. Cathepsin D has been found to be involved in the degradation of major tail tissues such as epidermis, muscle, spinal cord, notochord cells and blood cells in the regressing tail. Interestingly, it has also been found to be involved in the pre-regressing tail prior to visible tail regression. In addition, melanocytes have been described to be associated with degradation of different tail tissues.