Moving forward to understand the alteration of physiological mechanism by seed priming with different halo-agents under salt stress.

Soil salinity hampers the survival and productivity of crops. To minimize salt-associated damages in plant, better salt management practices in agriculture have become a prerequisite. Seed priming with different halo-agents is a technique, which improves the primed plant's endurance to tackle sodium. Salt tolerance is achieved in tolerant plants through fundamental physiological mechanisms- ion-exclusion and tissue tolerance, and salt-tolerant plants may (Na+ accumulators) or may not (Na+ excluders) allow sodium movement to leaves. While Na+ excluders depend on ion exclusion in roots, Na+ accumulators are proficient Na+ managers that can compartmentalize Na+ in leaves and use them beneficially as inexpensive osmoticum. Salt-sensitive plants are Na+ accumulators, but their inherent tissue tolerance ability and ion-exclusion process are insufficient for tolerance. Seed priming with different halo-agents aids in 'rewiring' of the salt tolerance mechanisms of plants. The resetting of the salt tolerance mechanism is not universal for every halo-agent and might vary with halo-agents. Here, we review the physiological mechanisms that different halo-agents target to confer enhanced salt tolerance in primed plants. Calcium and potassium-specific halo-agents trigger Na+ exclusion in roots, thus ensuring a low amount of Na+ in leaves. In contrast, Na+-specific priming agents favour processes for Na+ inclusion in leaves, improve plant tissue tolerance or vacuolar sequestration, and provide the greatest benefit to salt-sensitive and sodium accumulating plants. Overall, this review will help to understand the underlying mechanism behind plant's inherent nature towards salt management and its amelioration with different halo-agents, which helps to optimize crop stress performance.

Salmonella enterica serovars linked with poultry in India: antibiotic resistance profiles and carriage of virulence genes.

Salmonella is an important poultry pathogen with zoonotic potential. Being a foodborne pathogen, Salmonella-contaminated poultry products can act as the major source of infection in humans. In India, limited studies have addressed the diversity of Salmonella strains of poultry origin. This study represented 26 strains belonging to Salmonella serovars Typhimurium, Infantis, Virchow, Kentucky, and Agona. The strains were tested for resistance to 14 different antimicrobial agents using the Kirby-Bauer disk-diffusion assay. The presence of the invA, hilA, agfA, lpfA, sopE, and spvC virulence genes was assessed by polymerase chain reaction (PCR), and the genetic diversity was assessed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR). The highest resistance to tetracycline (n = 17; 65.38%) followed by nalidixic acid (n = 16; 61.53%) was detected among the strains. Among the strains (n = 17) phenotypically resistant to tetracycline, 94% (n = 16) were also positive for the tetA gene. Based on the presence of virulence genes, the strains were characterized into three virulence profiles (PI, P2, and P3). Among the investigated virulence genes, invA, hilA, agfA, and lpfA were present in all strains. The sopE gene was mostly associated with serovars Virchow (n = 3; 100%) and Typhimurium (n = 8; 80%), whereas spvC gene was exclusive for two Typhimurium strains that lacked sopE gene. ERIC-PCR profiling indicated clusters correlating their serovar, geographical, and farm origins. These results demonstrate that Salmonella isolates with a wide genetic range, antibiotic resistance, and virulence characteristics can colonize poultry. The presence of such strains is crucial for both food safety and public health.

A systematic review of the evaluation of endocrine-disrupting chemicals in the Japanese medaka (Oryzias latipes) fish.

Japanese medaka (Oryzias latipes) is an acceptable small laboratory fish model for the evaluation and assessment of endocrine-disrupting chemicals (EDCs) found in the environment. In this research, we used this fish as a potential tool for the identification of EDCs that have a significant impact on human health. We conducted an electronic search in PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (https://scholar.google.com/) using the search terms, Japanese medaka, Oryzias latipes, and endocrine disruptions, and sorted 205 articles consisting of 128 chemicals that showed potential effects on estrogen-androgen-thyroid-steroidogenesis (EATS) pathways of Japanese medaka. From these chemicals, 14 compounds, namely, 17ß-estradiol (E2), ethinylestradiol (EE2), tamoxifen (TAM), 11-ketotestosterone (11-KT), 17ß-trenbolone (TRB), flutamide (FLU), vinclozolin (VIN), triiodothyronine (T3), perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA), terephthalic acid (TPA), trifloxystrobin (TRF), ketoconazole (KTC), and prochloraz (PCZ), were selected as references and used for the identification of apical endpoints within the EATS modalities. Among these endpoints, during classification, priorities are given to sex reversal (masculinization of females and feminization of males), gonad histology (testis-ova or ovotestis), secondary sex characteristics (anal fin papillae of males), plasma and liver vitellogenin (VTG) contents in males, swim bladder inflation during larval development, hepatic vitellogenin (vtg) and choriogenin (chg) genes in the liver of males, and several genes, including estrogen-androgen-thyroid receptors in the hypothalamus-pituitary-gonad/thyroid axis (HPG/T). After reviewing 205 articles, we identified 108 (52.68%), 46 (22.43%), 19 (9.26%), 22 (17.18%), and 26 (12.68%) papers that represented studies on estrogen endocrine disruptors (EEDs), androgen endocrine disruptors (AEDs), thyroid endocrine disruptors (TEDs), and/or steroidogenesis modulators (MOS), respectively. Most importantly, among 128 EDCs, 32 (25%), 22 (17.18%), 15 (11.8%), and 14 (10.93%) chemicals were classified as EEDs, AEDs, TEDs, and MOS, respectively. We also identified 43 (33.59%) chemicals as high-priority candidates for tier 2 tests, and 13 chemicals (10.15%) show enough potential to be considered EDCs without any further tier-based studies. Although our literature search was unable to identify the EATS targets of 45 chemicals (35%) studied in 60 (29.26%) of the 205 articles, our approach has sufficient potential to further move the laboratory-based research data on Japanese medaka for applications in regulatory risk assessments in humans.

De Sanctis-Cacchione Syndrome with Subdural Effusion: A Rare Case from India with Review of Literature.

De Sanctis-Cacchione syndrome (DCS) formerly known as xerodermic idiocy is characterised by cutaneous photosensitivity, microcephaly, mental retardation, short stature, hypogonadism, spasticity, peripheral neuropathy and sensorineural deafness. Here in, we present the case of a four and half years old male child with features of severe acute malnutrition (SAM) with a typical bird like facies and sunken eyes who had history of photosensitive pruritic pigmentary skin lesions on sun exposed areas from a very early age of six months. Gross developmental delay, ataxia, microcephaly, short stature, hypogonadism and cachectic wasting were identified on examination and hypertransaminasemia and hypothyroidism were recorded from biochemical profile. Subsequent visual evoked response and brainstem evoked response audiometry revealed anterior visual pathway dysfunction and bilateral profound sensorineural hearing loss. Magnetic resonance imaging of brain yielded subdural effusion with mass effect in addition to cerebro-cerebral atrophy and demyelination. Skin biopsy further detected dysplastic changes and early signs of squamous cell carcinoma (SCC). Although few cases are reported sporadically throughout the world, to our best of knowledge till date only 11 such cases have been reported completely in Indian medical literature which makes our case report the 12th one with distinctive novel association of subdural effusion.

Experimental datasets on the immunohistological assessment of δ-cells in the islet organs of the endocrine pancreas of Japanese medaka (Oryzias latipes) fish exposed to graphene oxide.

The datasets of this article present the experimental parameters resulting from the assessment of δ-cells in the islet organs of the endocrine pancreas as a potential biomarker of endocrine disruption (ED) mediated by graphene oxide (GO), using Japanese medaka fish as the model. These datasets support the article "Evaluation of pancreatic δ-cells as a potential target site of graphene oxide toxicity in Japanese medaka (Oryzias latipes) fish". GO used in the experiments was either obtained from a commercial source or synthesized in the laboratory by us. GO was sonicated for 5 min in ice temperature before application. The experiments were conducted on reproductively active adult fish maintained as a breeding pair (one male and one female) in 500 ml balanced salt solution (BSS) either by immersion (IMR) in GO (20 mg/L) continuously for 96 h with the refreshing of media once in every 24 h, or by a single intraperitoneal (IP) administration of GO (100 µg/g) to both male and female partners. Control fish were maintained in BSS only (IMR experiment), or nanopure water (vehicle) was injected into the peritoneal cavity (IP experiment). The IP experimental fish were anesthetized in MS-222 (100 mg/L in BSS); the injected volume (0.5 µL/10 mg fish) never exceeds 50 µl/fish. After injection, the injected fish were allowed for recovery in clean BSS and after recovery both partners were transferred to 1 L glass jars with 500 mL BSS. During depuration, the media of the breeders refreshed once every 24 h and the eggs were collected. After 21 days, the survived fish were anaesthetized, and the trunk region was preserved in 4% paraformaldehyde in PBS (20 mM) containing 0.05% Tween 20. The phenotypic sex of adult fish was assessed externally by secondary sex characters (fin features) and internally by gonad (testis and ovary) histology. Once the location of pancreas was determined after HE stains, immunohistochemical technique was applied on next few slides using rabbit derived polyclonal antisomatostatin antibody as primary antibody and a commercial kit for colorimetric determination of δ-cells in the islet organs was used. Images were captured using an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. Using imagej software, a minimum 3 images of principal islets and one image of secondary islets were assessed. The immunoreactivity of δ-cells, due to neuron-like appearance and filopodia like processes, enabled us to separate them from other cell types found in the pancreatic islets of medaka. Based on immunoreactivity, we have classified islet cells into three categories; noncommunicating delta cells (NCDC), communicating cells (CC), and non-delta cells (NDC), and expressed as number of cells (NCDC/CC/NDC)/mm2 of islet organs. The nuclear area (µm2) and the linear length of filopodia of NCDCs were also considered for evaluation. Numerical data were analysed by Kruskal-Wallis test followed by Mann-Whitney's test as post hoc test and presented as means  ±  SEM. Statistically significant differences were considered for p ≤ 0.05.

Seed priming with NaCl helps to improve tissue tolerance, potassium retention ability of plants, and protects the photosynthetic ability in two different legumes, chickpea and lentil, under salt stress.

MAIN CONCLUSION: Seed priming with NaCl mimicked the conditions of natural priming to improve the tissue tolerance nature of sensitive legumes, which helps to maintain survivability and yield in mildly saline areas. Seed priming with NaCl is a seed invigoration technique that helps to improve plant growth by altering Na+ and K+ content under salt stress. Legumes are overall sensitive to salt and salinity hampers their growth and yield. Therefore, a priming (50 mM NaCl) experiment was performed with two different legume members [Cicer arietinum cv. Anuradha and Lens culinaris cv. Ranjan] and different morpho-physiological, biochemical responses at 50 mM, 100 mM, and 150 mM NaCl and molecular responses at 150 mM NaCl were studied in hydroponically grown nonprimed and primed members. Similarly, a pot experiment was performed at 80 mM Na+, to check the yield. Tissue Na+ and K+ content suggested NaCl-priming did not significantly alter the accumulation of Na+ among nonprimed and primed members but retained more K+ in cells, thus maintaining a lower cellular Na+/K+ ratio. Low osmolyte content (e.g., proline) in primed members suggested priming could minimize their overall osmolytic requirement. Altogether, these implied tissue tolerance (TT) nature might have improved in case of NaCl-priming as was also reflected by a better TT score (LC50 value). An improved TT nature enabled the primed plants to maintain a significantly higher photosynthetic rate through better stomatal conductance. Along with this, a higher level of chlorophyll content and competent functioning of the photosynthetic subunits improved photosynthetic performance that ensured yield under stress. Overall, this study explores the potential of NaCl-priming and creates possibilities for considerably sensitive members; those in their nonprimed forms have no prospect in mildly saline agriculture.

Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India.

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63-1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

Evaluation of pancreatic δ- cells as a potential target site of graphene oxide toxicity in Japanese medaka (Oryzias latipes) fish.

In continuation to our previous investigations on graphene oxide (GO) as an endocrine disrupting chemical (EDC), in the present experiment, we have investigated endocrine pancreas of Japanese medaka adults focusing on δ-cells in the islet organs as an endpoint. Breeding pairs of adult male and female fish were exposed to 0 mg/L (control) or 20 mg/L GO by continuous immersion (IMR) for 96 h, or to 0 µg/g or 100 µg/g GO by a single intraperitoneal (IP) administration and depurated 21 days in a GO-free environment. Histological investigations indicated that the endocrine cells are concentrated in one large principal islet, and several small secondary islets scattered within the mesentery near the liver and intestine. The cells of the islet organ are in various shapes with basophilic nuclei and eosinophilic cytoplasm. Immunohistochemical evaluation using rabbit polyclonal antisomatostatin antibody indicated that immunoreactivity is localized either at the periphery or at the central region in principal islets, and throughout the secondary islets, and found to be enhanced in fish exposed to GO than controls. The soma of δ-cells exhibits neuron-like morphology and have filopodia like processes. Cell sorting as non-communicating δ-cells (NCDC), communicating cells (CC), and non- δ-cells (NDC) indicated that within an islet organ, the population of NDCC is found to be the least and NDC is the highest. Our data further indicated that GO-induced impairments in the islet organs of medaka pancreas are inconsistent and could be affected by the exposure roots as well as the sex of the fish.

Experimental data-sets on the histopathological and immunohistological assessment of the Interrenal gland (adrenal homolog) of Japanese medaka (Oryzias latipes) fish exposed to graphene oxide.

The datasets of this article present the experimental parameters resulting from the assessment of adrenal gland as a potential biomarker of endocrine disruption mediated by graphene oxide (GO), a nanocarbon, using Japanese medaka fish as the model. These data sets support the article "Histopathological evaluation of the interrenal gland (adrenal homolog) of Japanese medaka (Oryzias latipes) exposed to graphene oxide". The experiments were conducted on reproductively active adult fish maintained as a breeding pair (one male and one female) in 500 mL balanced salt solution (BSS) either by immersion in GO (20 mg/L in BSS) continuously for 96 h with refreshing of media once in every 24 h or by a single intraperitoneal (IP) injection of GO (100 µg/g) to both male and female fish. The experimental fish were allowed breeding and assessed after 21 days post-treatment. Moreover, one day-post hatch (dph) Japanese medaka fries (orange-red variety) were exposed to different concentrations of GO (2.5-20 mg/L) by immersion in embryo-rearing medium (ERM) for 96 h (1-5 dph) with refreshing of media every 24h. Food was given to the adults, however, the larvae remained fasting during the GO-exposure (0-5 dph) period. Control adults and larvae were identically maintained either in BSS (adults) or ERM (larvae), with no GO. After treatment, both adults and the larvae were maintained in BSS with feeding in a GO-free environment. After 21 days post-treatment, adults, and after six weeks post-treatment, larvae, were anaesthetized in MS-222, and the trunk region was preserved in 4% paraformaldehyde in PBS (20 mM) containing 0.05% Tween 20. Evaluation of interrenal gland (IRG) in kidneys were made in 5 µm thick sections stained on haematoxylin-eosin (HE). The phenotypic sex of adults was assessed by secondary sex characters (fin features) and gonad (testis and ovary) histology; in larvae, phenotypic sex was determined by gonad histology and the genotypic sex by genotyping dmy gene. The location of IRG in the kidney were determined by immunohistochemical technique using rabbit polyclonal antityrosine hydroxylase antibody as primary antibody. The digital images of sections were captured using an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. Using imagej software, a minimum of 3 images of kidney consisting IRG were assessed for cell (separated as dark and pale stained nucleus after HE staining) sorting (cells/ mm2) and also measured the nuclear area (µm2). Counting of IRG cells, lined between the cardinal vein and the interstitial cells in the kidneys, were limited to maximum three layers in a given area. Numerical data, presented as means ± SEM, were analysed by one-way ANOVA followed by post-hoc Tukey's multiple comparison test or unpaired parametric 't' test including Welch's correction, if distributed normally; or by Kruskal-Wallis test followed by Mann-Whitney's test as post hoc test, if the data did not meet the criteria of using a parametric test. Statistically significant difference were considered for p ≤ 0.05. The collected data on IRG of Japanese medaka fish will be used for the assessment of GO as an EDC disposed in the environment.

Histopathological evaluation of the interrenal gland (adrenal homolog) of Japanese medaka (Oryzias latipes) exposed to graphene oxide.

Due to unique physicochemical properties and wide industrial and biomedical applications, graphene oxide (GO) is ubiquitous in the aquatic ecosystem. Using Japanese medaka (Oryzias latipes) fish as a model, we previously demonstrated minimal endocrine disrupting (ED) effects of GO on reproductive organs, and thyroids. Current study investigated the ED-effects of GO on the interrenal gland (IRG) of medaka. Breeding pairs of adult male and female fish were exposed to 0 mg/L (control) or 20 mg/L GO by continuous immersion for 96 h, or to 0 or 100 µg/g GO by intraperitoneal administration. Also, 1 day post-hatch (dph) larvae were exposed to different concentrations of GO (2.5-20 mg/L) for 96 h. IRG was evaluated by immunohistochemical techniques after 21 days depuration in adults and 6 weeks in larvae. IRG cells were counted and the nuclear area was measured in hematoxylin-eosin stained sections using ImageJ software. We found that IRG is distributed adjacent to the posterior cardinal vein and its branches within the head kidney. Columnar/oval shaped periodic acid-Schiff negative, tyrosine hydroxylase positive cells are arranged either in a single, or in groups, sometimes encircling a sinusoid, or in a straight chord, laying adjacent to the endothelium of the cardinal vein, and having eosinophilic cytoplasm with round/oval basophilic nuclei. GO effect on nuclei and cell population in IRG was inconsistent; depending on exposure route, sex, and/or age of the fish. Also, because of its high adsorptive property and sharp edges, GO probably agglomerated on IRG, and induced physical injury, and ED effects.

Influence of arsenate imposition on modulation of antioxidative defense network and its implication on thiol metabolism in some contrasting rice (Oryza sativa L.) cultivars.

Globally, many people have been suffering from arsenic poisoning. Arsenate (AsV) exposure to twelve rice cultivars caused growth retardation, triggered production of As-chelatin biopeptides and altered activities of antioxidants along with increase in ascorbate (AsA)-glutathione (GSH) contents as a protective measure. The effects were more conspicuous in cvs. Swarnadhan, Tulaipanji, Pusa basmati, Badshabhog, Tulsibhog and IR-20 to attenuate oxidative-overload mediated adversities. Contrastingly, in cvs. Bhutmuri, Kumargore, Binni, Vijaya, TN-1 and IR-64, effects were less conspicuous in terms of alterations in the said variables due to reduced generation of oxidative stress. Under As(V) imposition, the protective role of phytochelatins (PCs) were recorded where peaks height and levels of PCs (PC2, PC3 and PC4) were elevated significantly in the test seedlings with an endeavour to detoxify cells by sequestering arsenic-phytochelatin (As-PC) complex into vacuole that resulted in reprogramming of antioxidants network. Additionally, scatter plot correlation matrices, color-coded heat map analysis and regression slopes demonstrated varied adaptive responses of test cultivars, where cvs. Bhutmuri, Kumargore, Binni, Vijaya, TN-1 and IR-64 found tolerant against As(V) toxicity. Results were further justified by hierarchical clustering. These findings could help to grow identified tolerant rice cultivars in As-prone soil with sustainable growth and productivity after proper agricultural execution.

Comparative study on the influence of silicon and selenium to mitigate arsenic induced stress by modulating TCA cycle, GABA, and polyamine synthesis in rice seedlings.

Arsenic contamination of groundwater is a major concern for its usage in crop irrigation in many regions of the world. Arsenic is absorbed by rice plants mainly from arsenic contaminated water during irrigation. It hampers growth and agricultural productivity. The aim of the study was to mitigate the toxic effects of arsenate (As-V) [25 µM, 50 µM, and 75 µM] by silicon (Si) [2 mM] and selenium (Se) [5 µM] amendments on the activity of the TCA cycle, synthesis of γ-aminobutyric acid (GABA) and polyamines (PAs) in rice (Oryza sativa L. cv. MTU-1010) seedlings and to identify which chemical was more potential to combat this threat. As(V) application decreased the activities of tested respiratory enzymes and increased the levels of organic acids (OAs) in the test seedlings. Application of Si with As(V) and Se with As(V) increased the activities of respiratory enzymes and the levels of OAs. The effects were more pronounced during Si amendments. The activities of GABA synthesizing enzymes along with accumulation of GABA were increased under As(V) stress. During joint application of Si with As(V) and Se with As(V) the activity and the level of said parameters were decreased that indicating defensive role of these chemicals to resist As(V) toxicity in rice and Si amendments showed greater potential to reduce As(V) induced damages in the test seedlings. PAs trigger tolerance mechanism against As(V) in plants. PAs such as putrescine, spermidine and spermine were synthesized more during Si and Se amendments in As(V) contaminated rice seedlings to combat the toxic effects of As(V). Si amendments substantially modulated the toxic effects caused by As(V) over Se amendments in the As(V) challenged test seedlings. Thus, in future application of Si enriched fertilizer will be beneficial to grow rice plants with normal vigor in arsenic contaminated soil.

Selenium alleviates arsenic induced stress by modulating growth, oxidative stress, antioxidant defense and thiol metabolism in rice seedlings.

This study aims to investigate the potentiality of selenium in modulating arsenic stress in rice seedlings. Arsenate accumulation along with its transformation to arsenite was enhanced in arsenate exposed seedlings. Arsenite induced oxidative stress and severely affected the growth of the seedlings. Arsenate exposure caused an elevation in ascorbate and glutathione levels along with the activities of their metabolizing enzymes viz., ascorbate peroxidase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase. Phytochelatins content was increased under arsenic stress to subdue the toxic effects in the test seedlings. Co-application of arsenate and selenate in rice seedlings manifested pronounced alteration of oxidative stress, antioxidant defense, and thiol metabolism as compared to arsenate treatment only. ANOVA analysis (Tukey's HSD test) demonstrated the relevance of using selenate along with arsenate to maintain the normal growth and development of rice seedlings. Thus, exogenous supplementation of selenium will be a beneficial approach to cultivate rice seedlings in arsenic polluted soil.

Arsenic toxicity in the environment is a global concern, causes chronic signs of poisoning to plants and humans, leads to ecological imbalance. Selenium is known for its antagonistic characteristics and has been found to be effective in combating the adversities of arsenic at low concentrations (5 µM). The present study was performed to explore the comparative responses of rice seedlings during the joint application of selenium and arsenic in terms of growth, generation of oxidative stress, antioxidant defense, and thiol metabolism. Although the molecular basis of arsenic­selenium interaction is widely known a small number of reports were listed about the physio-chemical role of selenium against arsenic stress. Thus, we investigated the influence of selenium to alleviate arsenic-induced toxic effects by modulating the activities of antioxidant enzymes and reducing the levels of oxidative stress markers. It has been noted that selenium regulates thiol metabolism which is known to play a key role in growth preservation by restriction of arsenic translocation. The outcome from the study would be useful in field trials for sustainable agriculture in arsenic-contaminated soil.

Histological and Histochemical Evaluation of the Effects of Graphene Oxide on Thyroid Follicles and Gas Gland of Japanese Medaka (Oryzias latipes) Larvae.

Graphene oxide (GO) has become a topic of increasing concern for its environmental and health risks. However, studies on the potential toxic effects of GO, especially as an endocrine disrupting chemical (EDC), are very limited. In the present study we have used Japanese medaka fish as a model to assess the endocrine disruption potential of GO by evaluating its toxic and histopathologic effects on thyroid follicles and the gas gland (GG) of medaka larvae. One day post-hatch (dph) starved medaka fries were exposed to GO (2.5, 5.0, 10.0, and 20 mg/L) for 96 h, followed by 6 weeks depuration in a GO-free environment with feeding. Larvae were sacrificed and histopathological evaluation of thyroid follicles and the GG cells were done microscopically. Different sizes of spherical/oval shape thyroid follicles containing PAS positive colloids, surrounded by single-layered squamous/cuboidal epithelium, were found to be scattered predominantly throughout the pharyngeal region near the ventral aorta. We have apparently observed a sex-specific difference in the follicular size and thyrocytes height and a non-linear effect of GO exposure on the larvae on 47th day post hatch (dph). The GG is composed of large uniform epithelial cells with eosinophilic cytoplasm. Like thyroids, our studies on GG cells indicate a sex-specific difference and GO exposure non-linearly reduced the GG cell numbers in males and females as well as in XY and XX genotypes. Our data further confirm that sex effect should be carefully considered while assessing the toxicity of EDCs on the thyroid gland.

Comparative study of silicon and selenium to modulate chloroplast pigments levels, Hill activity, photosynthetic parameters and carbohydrate metabolism under arsenic stress in rice seedlings.

Arsenic (As) in groundwater severely harms global economic development by affecting growth and productivity of agricultural crops that causes human health risk. The comparative influence of silicon (Si) and selenium (Se) to modulate pigments levels, photosynthetic parameters using LI-6400XT Portable Photosynthesis System and carbohydrate metabolism under arsenate (As-V) stress in rice cv. MTU-1010 were evaluated. As(V) stress significantly decreased chlorophyll-a (32% on an average), chlorophyll-b (58% on an average), total chlorophyll (46% on an average), fluorescence intensity (31% on an average), carotene (39% on an average), xanthophyll (33% on an average), Hill activity (47% on an average) and the photosynthetic parameters, viz. intercellular CO2 concentration (52% on an average), net photosynthesis (54% on an average), transpiration rate (36% on an average) and stomatal conductance (38% on an average) in the test seedlings. As(V) + Si treatments enhanced the stated occurrences more than As(V) + Se treatments in rice seedlings. Sugar contents, viz. reducing (85% on an average) and non-reducing sugar (61% on an average), were increased, but starch content (57% on an average) was decreased in only As(V)-treated rice seedlings. The activities of carbohydrate metabolizing enzymes were increased, while sucrose synthase activity was decreased due to As(V) toxicity in the test seedlings. Co-application of Si and As(V) as well as Se and As(V) showed ameliorative effects on sugar and starch contents along with the activities of carbohydrate metabolizing enzymes, but more potential effect was observed under combined application of Si and As(V) in rice seedlings. Thus, it is an important purpose of this paper to compare the ability of Se and Si to alleviate As(V) toxicity in rice seedlings which will be an effective approach to develop possible strategies in As-contaminated agricultural soil to improve normal growth and productivity of rice plants.

Experimental data sets on the evaluation of graphene oxide as a thyroid endocrine disruptor and a modulator of gas gland cells in Japanese medaka (Oryzias latipes) larvae at the onset of maturity.

This article presents the experimental datasets obtained from the histological/histochemical studies of endocrine disrupting effects of graphene oxide (GO) on thyroid follicles and gas gland (GG) cells of Japanese medaka larvae at the onset of maturity. The experiment was conducted on one day-post hatch (dph) starved fries (orange-red variety) immersed in different concentrations of GO (2.5-20.0 mg/L) and no GO (controls) in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). After treatment, larvae were maintained in balanced salt solution (BSS) with food and allowed depuration for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS 222 and their total lengths (mm) and weights (mg) were measured, and they were then cut into three small pieces (head, trunk, and tail). Head and trunk regions were fixed in 4% PFA in 20 mM PBS for 48 h at room temperature and the post-anal tail was preserved in TRI reagent and kept at -20 °C until analysis. Tissues in 4% PFA were used for cutting 5µm thick paraffin sections in a manual rotary microtome. Sections of head regions were evaluated for thyroid follicles after hematoxylin-eosin (HE) or Periodic acid-Schiff (PAS) staining. Trunk sections were used for swim bladder (SB) inflation studies and for phenotypic sex (ovary and testis) of the larvae after HE staining. Genetic sex assessment was made from tail DNA by genotyping Y chromosome-specific male sex-determining gene dmy. Digital images were captured by using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 microscope with DP22 camera and CellSens software. Images of thyroid follicles and GG cells were analyzed using imagej software. HE stained histological sections of thyroid follicles near the heart and branchial regions were captured and the area (µm2) of individual follicles (minimum 3) available in the entire section were measured. The heights of thyrocytes (µm) were determined directly. Manual counting of GG cells was made from the digital images captured in several regions of the SB avoiding blood cells and other cells which have indistinct nucleus and pale cytoplasm; results were expressed as the number of GG cells/mm2. Data were analyzed by GraphPad prism version 7.04. For normally distributed data, one-way ANOVA followed by post-hoc Tukey's test or unpaired parametric "t" test including Welch's correction was used. Otherwise, Kruskal-Wallis test followed by nonparametric Mann-Whitney's test as a post hoc test was used. Data were expressed as means ±SEM and the level of significance was set at p < 0.05.

Experimental data-sets on sex reversal and histopathological assessment of potential endocrine disrupting effects of graphene oxide on Japanese medaka (Oryzias latipes) larvae at the onset of maturity.

The datasets of this article present the experimental parameters resulting from the assessment of sex reversal (SR) as a biomarker of endocrine disrupting effect of graphene oxide (GO), together with the histopathological assessment of ovary, testis, liver and kidneys of medaka larvae. These data sets support the published article "Sex-reversal and histopathological assessment of potential endocrine-disrupting effect of graphene oxide on Japanese medaka (Oryzias larvae) larvae." The experiments were conducted on one day-post hatch (dph) Japanese medaka fries (orange-red variety) exposed to different concentrations of GO (2.5-20 mg/L) by immersion in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). No food was given during the GO-exposure period. Controls (no GO) were identically maintained in ERM. After treatment, the larvae were maintained in balanced salt solution (BSS) with feeding and allowed to grow for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS-222, and the total length (mm) and body weight (mg) were recorded. For histopathological and phenotypic sex assessments, after sacrifice, the body excluding post-anal tail was preserved in 4% paraformaldehyde containing 0.05% Tween 20; ovary, testis, liver and kidneys were evaluated in 5 µm thick sections stained on haematoxylin eosin (HE) following OECD guidelines. The photomicrographs of sections were made using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 inverted microscope with DP22 camera and CellSens software. A minimum 3 images of gonads in different regions were further analysed by imagej software and used for counting spermatogonia (SPG) and spermatocytes (SPT) in testis as well as perinucleolar (PNO) and cortical alveolar (CAO) oocytes in ovary. Data were expressed as number of SPG or SPT/mm2 testis and % CAO or PNO in an ovary. Preserved tail in TRI reagent was used for genomic DNA extraction and the genetic sex was assessed by genotyping Y chromosome-specific male sex-determining gene dmy. Two different sets of buffers and primers were used and the reactions were conducted in a thermal cycler. The amplified products were separated in 2% agarose gel containing 0.01% ethidium bromide. The gels were viewed on an UV illuminator and the genotypes were identified by visual inspection. The first primer set amplified a 355 bp product for XY genotypes and no amplification for XX. The second set of primers amplified two products; one at 1249 bp and another at 986 bp for XY, and one product at 1249 bp for XX. Experimental data were expressed as means ± SD or SEM, analysed either by one-way analysis of variance (ANOVA) followed by post-hoc Tukey's multiple comparison test or unpaired parametric 't' test including Welch's correction, if distributed normally (lengths and weights), or by Kruskal-Wallis test followed by Man-Whitney's test as post hoc test, if data (stromal follicles in ovary and SPGs and SPTs in testis) did not meet the criteria of using a parametric test. Statistically significant difference were considered for p ≤ 0.05.

Silicon augments salt tolerance through modulation of polyamine and GABA metabolism in two indica rice (Oryza sativa L.) cultivars.

Polyamines (PA) have multifarious roles in plant-environment interaction and stress responses. In conjunction with GABA shunt, they regulate induction of tolerance under salinity stress in plants. Here, we tested the hypothesis that silicon improves salt tolerance through mediating vital metabolic pathways rather than acting as a mere mechanical barrier. Seedlings of two rice (Oryza sativa L.) cultivars MTU 1010 (salt-sensitive) & Nonabokra (salt-tolerant) growing in hydroponic culture were treated with NaCl (0, 25, 50 & 100 mM) combined with or without Si (2 mM). NaCl stress enhanced PA synthesizing enzymes activity and PA production in salt tolerant cultivar Nonabokra, whereas in the sensitive cultivar, MTU 1010 both declined. Enhanced activities of GABA synthesizing enzymes along with a decline in the activities of GABA degrading enzymes under NaCl exposure led to GABA accumulation in both the cultivars. The interactive effects of silicon and NaCl also induced the activities of the enzymes related to polyamine biosynthesis and inhibited polyamine degrading enzymes that enhanced PA contents in the cultivars. Supplemental Si decreased endogenous GABA levels by modulating GABA metabolising enzymes under NaCl stress. On the basis of all tested parameters cv. MTU 1010 was proven to be more responsive towards silicon application than cv. Nonabokra. Such study of silicon-induced polyamine accretion and reduced GABA accumulation may lower oxidative damage in rice cultivars under NaCl stress and thereby form a successful strategy to boost tolerance.

Sex-reversal and Histopathological Assessment of Potential Endocrine-Disrupting Effects of Graphene Oxide on Japanese medaka (Oryzias latipes) Larvae.

Sex-ratio is considered as an end point during endocrine disrupting chemicals (EDCs) evaluation. Many fish species including Japanese medaka have XX/XY sex determination mechanism, however, sex reversal (SR) can be induced by external and genetic factors. SR imposed an imbalance in natural sex ratio of a population living in any ecosystem. Considering SR as an end point, we aimed to investigate the potential EDC effects of graphene oxide (GO), a nanocarbon, using Japanese medaka as a model. One-day post-hatch (dph) medaka fries were exposed to GO (2.5, 5.0, 10.0 and 20 mg/L) for 96 h without food, followed by 6 weeks depuration in a GO-free environment with feeding. Phenotypic sex was determined by gonad histology; genotypic sex by genotyping Y-chromosome-specific male sex determining gene, dmy. Our data indicated testes in both XY and XX genotypes, while ovaries were only in XX females. Histopathology of XY and XX testis showed isogenic spermatocysts with active spermatogenesis. Distribution of spermatocytes (SPTs), not the spermatogonium (SPGs), showed enhancement in XY than XX testis. Female phenotypes had single ovary, either in stage 0 or 1. Ovo-testis/testis-ova were absent in XX or XY gonads. GO (2.5-20 mg/L) had inconsistent concentration-dependent effect in both SPGs and SPTs; however, no effect on ovarian follicles. Despite genotypic differences (XY/XX), in the histopathology/histochemistry of liver and kidneys GO effects was found to be minimum. Taken together, present study showed spontaneous induction of SR in some XX genotypes; however, exposure of fasting fries to GO had no apparent EDC effects.

Experimental datasets on the characterization of graphene oxide and its reproductive and developmental effects on Japanese medaka (Oryzias latipes) fish.

The datasets of this article present the experimental parameters resulting from the synthesis and characterization of graphene oxide (GO) using scanning and transmission electron microscopy (SEM, TEM) and spectrophotometric (FTIR, AFM, EDX) methods, and the assessment of its toxicological and endocrine-disrupting effects on the Japanese medaka fish by acute toxicity testing, and histopathological evaluations. These datasets support the article "Reproductive and Developmental Effects of Graphene Oxide on Japanese Medaka (Oryzias latipes)". GO synthesis was performed following the modified Hummer's method. Its particle diameter and zeta potential were determined using Zeta Sizer Nano ZS analyzer, and characterized by SEM and TEM. After 5 min sonication in water, GO (25-200 µg/g) was injected intraperitoneally to the reproductively active male and female fish maintained as a breeding pair (one male, one female) in 500 mL balanced salt solution (BSS) in glass jars under standard laboratory conditions (25±1 °C; 16L:8D light cycle). The control fish were injected with water. The maximum volume of the injected material is 1 µL/10 mg body weight. To avoid movement, during injection the fish were briefly anesthetized in MS 222 (100 mg/L) and after injection transferred to BSS for recovery. LD50 values of GO related to fish mortality were determined from the linear regression analysis using a software program. Reproductive activities (fecundity) were determined by daily collection of eggs 7 days before and 21 days after injection from a breeding pair and expressed as percent eggs laid every day post-injection relative to the average (mean of 7 days) eggs laid prior to injection. Developmental abnormalities of the embryos were assessed by culturing the collected fertilized eggs in ERM for a maximum period of 14 day-post fertilization (dpf). The fish that survived after 21days post-injection were sacrificed and the entire fish excluding post-anal tail were cut into three small pieces and fixed in 4% paraformaldehyde containing 0.05% Tween 20. Histopathological evaluations of gonads (ovary and testis), liver, and kidneys were made in 5 µm thick sections stained mainly on hematoxylin and eosin (HE) following the guidelines published by OECD. The Photomicrographs of the sections were made using Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or in Nikon Eclipse 50i microscope attached to Nikon DS-Fi1 camera. Four types of follicles in the stromal compartments of the ovary, perinucleolar (PNO), cortical alveolar (CAO), early vitellogenic (EVO) and late vitellogenic (LVO) were considered as differentiating, and the post ovulatory and atretic follicles were considered as degenerating follicles, and counted in an entire section made through four different regions (anterior, upper middle, lower middle, and anal) of the ovary. The follicular data were expressed as percent follicles (individual follicles or differentiating or degenerating) or as the ratio of differentiating and degenerating follicles found in that particular region of the ovary. The data were analyzed either by one- or two-way ANOVA followed by post-hoc Tukey's multiple comparison test and expressed as means ±SEM.