Pre-clinical and cellular safety assessment of oral administered DHA rich microalgae oil from Schizochytrium sp. (Strain ATCC-20889): acute, sub-chronic and genotoxicity.

The lack of toxicity data for DHA-rich oil from Schizochytrium sp. (Strain ATCC-20889) leads to its exclusion from the Qualified Presumption of Safety list. Therefore, present study addresses toxicity evaluation of DHA-rich microalgae oil using ex-vivo (cytotoxicity assay) and in-vivo methods (acute (OECD 423 guidelines), sub-chronic (OECD 452 guidelines), and genotoxicity assay). The ex-vivo results showed >90% cell viability of Caco-2 cells after 48 h of treatment (200 µg/mL of DHA). Additionally, the in-vivo acute toxicity study found that microalgae oil was nontoxic and classified under category 5 molecule according to OECD 423 guidelines with a highest degree of safety at 2000 mg/kg b.w. The in-vivo sub-chronic study revealed no significant mortality and changes in feed intake, body weight, haematological, biochemical, neurological, and urine parameters after repeated 180-days administration of DHA-rich microalgae oil at 250 mg/kg, 500 mg/kg, and 1000 mg/kg. Moreover, histopathology evaluation, comet assay, chromosomal aberration, and micronuclei assay also confirmed the nontoxic behavior of DHA-rich oil. Thus, the results from the ex-vivo and in-vivo studies indicate that DHA-rich oil from Schizochytrium sp. (Strain ATCC-20889) is safe for use as a novel food, and can be included in infants, adults, pregnant women, and children formula.

Genotoxic and mutagenic potential of 7-methylxanthine: an investigational drug molecule for the treatment of myopia.

7-Methylxanthine (7-MX, CAS No. 552-62-5, purity 99.46%) is the first orally administered drug candidate, which showed anti-myopic activity in different pre-clinical studies. In the present study, we investigated the in-vivo genotoxic and mutagenic toxicity of 7-MX in Wistar rats using comet/single-cell gel electrophoresis, chromosomal aberration and micronucleus assays after oral administration. For the single-dose study (72 h), two doses of 7-MX 300 and 2000 mg/kg body weight were selected. For a repeated dose 28 d study, three doses (250, 500, and 1000 mg/kg) of 7-MX were selected. The doses were administered via oral gavage in the suspension form. Blood and major vital organs such as bone marrow, lung and liver were used to perform comet/single cell gel electrophoresis, chromosomal aberration, and micronucleus assays. The in-vitro Ames test was performed on TA98 and TA100 strains. In the chromosomal aberration study, a non-significant increase in deformities such as stickiness, ring chromosome, and endoreduplication was observed in bone marrow cells of 7-MX treated groups. These chromosomal alterations were observed upon treatment with doses of 2000 mg/kg single dose for 72 h and 1000 mg/kg repeated dose for 28 d. At a dose of 500 mg/kg, DNA damage in terms of tail length, tail moment, % tail DNA and the olive tail moment was also found to be non-significant in 7-MX treated groups. The Ames test showed the non-mutagenic nature of 7-MX in both strains of TA98 and TA100 of Salmonella typhimurium with or without metabolic activation. Thus, the present work is interesting in view of the non- genotoxicity and non-mutagenicity of repeated doses of 7-MX.

Teratogenicity and accumulation of triclosan in the early life stages of four food fish during the bioassay.

TCS [5-chloro-2-(2,4-dichlorophenoxy)phenol] caused a concentration dependent delay in embryonic development, delay and decline in hatching and reduction in length and weight of hatchlings along with an increase in abnormal/deformed embryos and larvae and percent mortality. These parameters varied in a species specific manner and increased with TCS residue in body. The 96 h LC50 values of TCS for Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala were estimated at 0.315, 0.116, 0.096 and 0.131 mg/L, respectively. Hatching got delayed by 16.33 h for C. carpio (0.47 and 0.50 mg/L TCS) and C. idella (0.20 mg/L TCS) but by 18.07 h for L. rohita (0.15 mg/L TCS) and by 19.33 h for C. mrigala (0.18 mg/L TCS). Spine malformations, oedema (yolk sac and cardiac) and deflated swim bladder were present in 100% larvae of C. carpio, C. idella, L. rohita and C. mrigala at 0.30, 0.08, 0.13 and 0.14 mg/L TCS, respectively. TCS also caused hemorrhage (all but C. idella, only 3.33%), albinism and deformed caudal fin (C. idella), hypopigmentation and rupturing of yolk sac (C. mrigala), gas bubble disease (C. mrigala and L. rohita), fusion of eyes (C. carpio) and degeneration of digestive tract (L. rohita) in 10-40% hatchlings. Exposed hatchlings were very weak and paralyzed, could not swim and remained settled at the bottom of jars. Embryonic development was observed to be an early indicator of the toxicity of TCS as oedema and bubbles in yolk were observed in 40-100% embryos/hatchlings at 0.08 mg/L TCS while 100% mortality was observed between 0.15 and 0.50 mg/L TCS. L. rohita was most sensitive and C. carpio was least sensitive to the stress of TCS. Accumulation of TCS in the hatchlings (1/10 of TCS in water) after 96 h exposure hints that even small quantities of TCS may change species diversity in natural waters.

Entropy Churn Metrics for Fault Prediction in Software Systems.

Fault prediction is an important research area that aids software development and the maintenance process. It is a field that has been continuously improving its approaches in order to reduce the fault resolution time and effort. With an aim to contribute towards building new approaches for fault prediction, this paper proposes Entropy Churn Metrics (ECM) based on History Complexity Metrics (HCM) and Churn of Source Code Metrics (CHU). The study also compares performance of ECM with that of HCM. The performance of both these metrics is compared for 14 subsystems of 5different software projects: Android, Eclipse, Apache Http Server, Eclipse C/C++ Development Tooling (CDT), and Mozilla Firefox. The study also analyses the software subsystems on three parameters: (i) distribution of faults, (ii) subsystem size, and (iii) programming language, to determine which characteristics of software systems make HCM or ECM more preferred over others.

Fish erythrocytes as biomarkers for the toxicity of sublethal doses of an azo dye, Basic Violet-1 (CI: 42535).

The aim of the present study was to investigate poikilocytosis in Labeo rohita (an important food fish) as an early indicator of stress due to an azo dye, Basic Violet-1 (CI: 42535). This dye was observed to be very toxic to test fish (96 h LC50 as0.45 mg/L dye). Fish were given short-term (96 h) and subchronic (150 days) exposures to the dye, and poikilocytosis was recorded under light and scanning electron microscopy (SEM). Light microscopy helped in identification of micronuclei along with irregularities, notches, blebs, lobes, crenation, clumps, chains, spherocytes, vacuolation, and necrosis in erythrocytes. However, SEM indicated shrinkage, oozing of cytoplasm, and several new abnormal shapes including marginal foldings, discocytes, keratocytes, dacrocytes, degmacytes, acanthocytes, echinocytes, protuberances, stomatocytes, drepanocytes, holes in the membrane, stippling/spicules, crescent-shaped cells, triangular cells, and pentagonal cells. Earlier studies speculated changes in the membrane to be responsible for clumping and chaining of erythrocytes, whereas the present SEM study clearly indicates that oozing out of cytoplasm is also responsible for the formation of chains and clumps. This study also shows that erythrocytes exhibit pathological symptoms before the appearance of other external symptoms such as abnormal behavior or mortality of fish. There was a dose- and duration-dependent increase; therefore, poikilocytosis, especially echinocytes, spherocytes, and clumps, can act as a biomarker for the stress caused by azo dyes.

Attenuation of mitochondrial refractory epilepsy in rotenone corneal kindling model of drug resistance by idebenone: An approach to bypass mitochondrial complex I.

OBJECTIVE: To assess the potential of bypassing mitochondrial complex I with idebenone to overcome drug resistance in a Rotenone corneal kindling (RCK) mouse model of mitochondrial refractory epilepsy. MATERIAL AND METHOD: Resistance was developed by administering rotenone 2.5 mg/kg intraperitoneally once and corneal kindling twice daily. The kindling development took 15 days, and pre-treatment resistance validation was carried out with five different antiseizure drugs: pregabalin, levetiracetam, valproate, lamotrigine, and phenytoin. The treatment drug, Idebenone (IDB) was given at doses of 10, 20, and 40 mg/kg intraperitoneally for 10 days. The post-treatment resistance validation was evaluated with same standard drugs in same order along with other parameters assessment, such as NAD(P)H: quinone oxidoreductase 1 (NQO1), ATP, GSH, and TBARS. RESULTS: The pre-treatment resistance validation shows an inability of standard drugs to attenuate seizure scores by rotenone kindling, justifying the development of drug resistance. IDB successfully abolished the resistance developed in RCK model. IDB elevated the levels of ATP and NQO1 and showed antioxidant activity by elevating GSH and attenuating TBARS. CONCLUSION & FUTURE DIRECTION: IDB have successfully elevated the level of ATP, NQO1 in RCK model, hence proving the complex I bypass hypothesis. Thus, IDB can be the drug of choice for mitochondrial epilepsies involving drug refractoriness as adjuvant with anticonvulsant drugs.

Ameliorative effect of diclofenac in rotenone corneal kindling model of drug-resistant epilepsy: Edge of dual COX and KMO inhibition.

Epilepsy affects millions of people worldwide, about one-third patients with epilepsy exhibits resistance to available antiseizures medications, known as drug-resistant epilepsy (DRE). Mitochondrial dysfunction has been implicated as a hallmark in drug-resistant epilepsy via activation of microglial kynurenine 3-monooxygenase (KMO) and cyclooxygenase (COX) enzymes, leading to neuroinflammation and oxidative stress. Diclofenac, an equipotent non selective cyclooxygenase inhibitor, has inhibitory action on KMO enzyme and has also shown anti-inflammatory and antioxidant properties in animal models of epilepsy. These properties make it a suitable candidate for amelioration of DRE. However, its potential in drug-resistant epilepsy remained unexplored till date. In this study, dose dependent effect of diclofenac (5 mg/kg, 10 mg/kg, 20 mg/kg) has been explored in rotenone corneal kindling model of mitochondrial DRE. The results of our study revealed the induction of drug resistance to antiseizure medications and induced kynurenine 3-monooxygenase activity in rotenone corneal kindled epileptic mice in comparison to naive mice. Treatment of rotenone corneal kindled epileptic mice with diclofenac resulted in a significant decrease in drug resistance to antiseizure medications as evident by a reduction in seizure score in the treatment groups as compared to control group, in post-treatment resistance validation. The kynurenine 3-monooxygenase inhibitory activity (as evidenced by decreased levels of neurotoxic quinolinic acid) and the antioxidant effect (as evident by significantly reduced oxidative stress) in the diclofenac treated groups, emerged as a major contributor for its ameliorative action. Findings of this study suggests, diclofenac can be used as an adjunct therapy in amelioration of drug-resistant epilepsy.

Strategic pathway analysis for dual management of epilepsy and comorbid depression: a systems biology perspective.

Depression is a common psychiatric comorbidity among patients with epilepsy (PWE), affecting more than a third of PWE. Management of depression may improve quality of life of epileptic patients. Unfortunately, available antidepressants worsen epilepsy by reducing the seizure threshold. This situation demands search of new safer target for combined directorate of epilepsy and comorbid depression. A system biology approach may be useful to find novel pathways/markers for the cure of both epilepsy and associated depression via analyzing available genomic and proteomic information. Hence, the system biology approach using curated 64 seed genes involved in temporal lobe epilepsy and mental depression was applied. The interplay of 600 potential proteins was revealed by the Disease Module Detection (DIAMOnD) Algorithm for the treatment of both epilepsy and comorbid depression using these seed genes. The gene enrichment analysis of seed and diamond genes through DAVID suggested 95 pathways. Selected pathways were refined based on their syn or anti role in epilepsy and depression. In conclusion, total 8 pathways and 27 DIAMOnD genes/proteins were finally deduced as potential new targets for modulation of selected pathways to manage epilepsy and comorbid depression. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00208-1.

Dynamic alterations in physiological and biochemical indicators of Cirrhinus mrigala hatchlings: A sublethal exposure of triclosan.

Triclosan (TCS), a biocide used in various day-to-day products, has been associated with several toxic effects in aquatic organisms. In the present study, biochemical and hematological alterations were evaluated after 14 d (sublethal) exposure of tap water (control), acetone (solvent control), 5, 10, 20, and 50 µg/L (environmentally relevant concentrations) TCS to the embryos/hatchlings of Cirrhinus mrigala, a major freshwater carp distributed in tropic and sub-tropical areas of Asia. A concentration-dependent increase in the content of urea and protein carbonyl, while a decrease in the total protein, glucose, cholesterol, triglycerides, uric acid, and bilirubin was observed after the exposure. Hematological analysis revealed a decrease in the total erythrocyte count, hemoglobin, and partial pressure of oxygen, while there was an increase in the total leucocyte count, carbon dioxide, and partial pressure of carbon dioxide and serum electrolytes. Comet assay demonstrates a concentration-dependent increase in tail length, tail moment, olive tail moment, and percent tail DNA. An amino acid analyzer showed a TCS-dose-dependent increase in various amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed different proteins ranging from 6.5 to 200 kDa, demonstrating TCS-induced upregulation. Fourier transform infrared spectra analysis exhibited a decline in peak area percents with an increase in the concentration of TCS in water. Curve fitting of amide I (1,700-1600 cm-1) showed a decline in α-helix and turns and an increase in ß-sheets. Nuclear magnetic resonance study also revealed concentration-dependent alterations in the metabolites after 14 d exposure. TCS caused alterations in the biomolecules and heamatological parameters of fish, raising the possibility that small amounts of TCS may change the species richness in natural aquatic habitats. In addition, consuming TCS-contaminated fish may have detrimental effects on human health. Consequently, there is a need for the proper utilisation and disposal of this hazardous compound in legitimate quantities.

Effectiveness of indigenous ready-to-use therapeutic food in community-based management of uncomplicated severe acute malnutrition: a randomized controlled trial from India.

A randomized controlled trial was conducted in Chandigarh, India (2011), to determine the effectiveness of indigenous ready-to-use therapeutic food (RUTF) in community-based management of uncomplicated severe acute malnutrition (SAM). Intervention was through outpatient therapeutic program site (OTP). Study and control group children (6 months-5 years) were followed up weekly for 12 weeks, in OTP and at home. All children received supplementary nutrition through anganwadis under integrated child development scheme. Study children, in addition, received therapeutic dose of RUTF in OTP. Primary outcome, 115% of baseline weight, was attained in 6 of 13 (46.2%) and 1 of 13 (7.7%) children among study and control group, respectively [odds ratio: 10.28, 95% confidence interval (CI): 1.02-103.95]. Compared with control group, addition of RUTF in study group resulted in average additional increase in weight by 13 g/kg of baseline weight/week/child (95% CI: 2-23). Indigenous RUTF was effective in community-based management of uncomplicated SAM.

Physiological, biochemical and structural changes in tomato plants by vermicompost application in different exposure periods under glass house conditions.

In modern era, various inorganic fertilizers and pesticides are used as plant growth supplements in a variety of crop in order to gain maximum output and also reported as hazardous to mankind as well as environment. In addition, some of the plants died in initial phase of the growth after germination due to poor nutrient content of the soil or exposure to biotic stresses. In the beginning of sustainable agriculture, these chemical fertilizers were replaced with some alternative growth boosters such as organic fertilizers. In the present study, vermicompost was prepared using garden waste and cattle dung followed by analysis of various physico-chemical properties. Then tomato seeds were allowed to germinate in soil and supplemented with different doses of vermicompost (0-100%). The plants were harvested after 10 and 45 days of their germination and tissues were subjected to analysis of various morphological and biochemical parameters. Morphological parameters included root length, shoot length, root fresh weight, shoot fresh weight and number of leaves. Whereas biochemical parameters such as protein content, antioxidative enzymes (catalase, superoxide dismutase, ascorbate peroxidase, polyphenol oxidase), non-enzymatic antioxidants (ascorbic acid, glutathione, tocopherol), osmolytes (proline, carbohydrate), photosynthetic pigments (chlorophyll, carotenoid) and secondary metabolites (phenol, flavonoid, anthocyanin) were estimated on UV-visible spectrophotometer using standard protocols. Further, structural analysis of plant tissue was done using fourier transform infrared spectroscopy spectra (FTIR) and carbon hydrogen nitrogen (CHN) elemental analyzer. Results obtained from the present study revealed significant difference in all morphological and biochemical markers at both 10 and 45 days intervals of time. Further, growth of all plants was found to be directly proportional to the concentration of vermicompost and exposure duration. FTIR spectra and CHN analyses reveal the breakdown of various complex compounds and their transformation from Vcom amended soil to roots of plants. This is the first study in which significant changes were observed in growth, physiology and structural composition of tomato plants at two different exposure periods (10 and 45 days) under glass house conditions which further concluded that vermicompost has a significant potential for increasing plant growth.

A network pharmacology approach to explore pharmacological mechanisms of Asparagus racemosus for ameliorative effect in epilepsy and comorbid progressive memory dysfunction.

Background: Network pharmacology approach has been observed a powerful tool to predict underlying complex pharmacological mechanism of herbs. Asparagus racemosus has been reported to show ameliorative effects in treating epilepsy and comorbid memory dysfunction but mechanism of this amelioration is elusive. Hence a network pharmacology approach was employed to investigate the plausible mechanism of A. recemosus. Methodology: : Bioactive compounds of A. racemosus were extracted based on the TCMSP, PCIDB, and BATMAN-TCM database. The potential targets of bioactive compounds were collected using target fishing. Epilepsy and comorbid dementia genes were collected from DISGENET. A PPI network among these targets was constructed using the intersecting key targets between herb targets and disease targets. Besides, DAVID bioinformatics resource was utilized for the pathway enrichment analysis on GO and KEGG. Ultimately, phytochemical compound-target genes-Pathways network has been assembled utilizing Cytoscape to decipher the mechanism of the herb. Results: The network analysis revealed that 5 targets (CASP3, TNF, VEGFA, PTGS2 and CNR1) might be the key therapeutic targets of asparagus on Epilepsy comorbid Alzheimer's disease. Based on high connectivity, four hub compounds with the highest connectivity were noted and it includes Shatavarin V, Sarsasapogenin, Shatavarin IX, and Shatavarin VI. A total of 19 KEGG terms were enriched as the potential pathways of A. racemosus in Epilepsy comorbid Alzheimer's disease. Conclusion: This study envisaged the pharmacological and molecular mechanism of A. racemosus against epilepsy comorbid Alzheimer's disease and put forward a strategy to uncover the mechanisms of Traditional Indian Medicine based on network pharmacology. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00169-x.

Adjunct antiseizure effect of clotrimazole in a rotenone corneal kindling mouse model of mitochondrial drug-resistant epilepsy.

This study aimed to investigate the therapeutic potential of clotrimazole, an inhibitor of the transient receptor potential cation channel, for treating mitochondrial drug-resistant epilepsy and to understand its underlying neurochemical mechanisms. Adult albino mice underwent rotenone-corneal kindling, receiving daily electric shocks (15 mA, 20 V, 6-Hz for 3 s) through a corneal electrode, to induce mitochondrial drug-resistant epilepsy. The onset of drug resistance was confirmed by the significant (p < 0.05) lack of seizure control with standard antiseizure medications including levetiracetam (40 mg/kg), valproate (250 mg/kg), phenytoin (35 mg/kg), lamotrigine (15 mg/kg), and carbamazepine (40 mg/kg). Drug-resistant mice were then classified into one vehicle-treated group and three groups treated with varying doses of clotrimazole (40, 80, and 160 mg/kg orally). Neurochemical analysis of the seizurogenic hippocampus and cerebral cortex was conducted using high-performance liquid chromatography with an electrochemical detector. Administration of clotrimazole alongside standard antiseizure medications led to a significant decrease (p < 0.05) in seizure scores suggesting the restoration of antiseizure effects. Neurochemicals, including tryptophan, serotonin, kynurenine, serine, taurine, gamma-aminobutyric acid, and glutamate, were significantly restored post-clotrimazole treatment. Overall, the present study underscores the adjunct antiseizure effect of clotrimazole in a rotenone corneal kindling mouse model of mitochondrial drug-resistant epilepsy, emphasising its role in neurochemical restoration.

Biopreservative efficacy of Enterococcus faecium-immobilised film and its enterocin against Salmonella enterica.

The growing awareness about the adverse health effects of artificial synthetic preservatives has led to a rapid increase in the demand for safe food preservation techniques and bio preservatives. Thus, in this study, the biopreservatives efficacy of enterocin-producing Enterococcus faecium Smr18 and its enterocin, ESmr18 was evaluated against Salmonella enterica contamination in chicken samples. E. faecium Smr18 is susceptible to the antibiotics penicillin-G, ampicillin, vancomycin, and erythromycin, thereby indicating that it is a nonpathogenic strain. Further, the enterocin ESmr18 was purified and characterised as a 3.8 kDa peptide. It possessed broad spectrum antibacterial activity against both Gram-positive and Gram-negative pathogens including S. enterica serotypes Typhi and Typhimurium. Purified ESmr18 disrupted the cell membrane permeability of the target cell thereby causing rapid efflux of potassium ions from L. monocytogenes and S. enterica. Chicken samples inoculated with S. enterica and packaged in alginate films containing immobilised viable E. faecium resulted in 3 log10 colony forming units (CFU) reduction in the counts of S. enterica after 34 days of storage at 7-8 °C. The crude preparation of ESmr18 also significantly (p < 0.05) reduced the CFU counts of salmonella-inoculated chicken meat model. Purified ESmr18 at the concentration upto 4.98 µg/ml had no cytolytic effect against human red blood cells. Crude preparation of ESmr18 when orally administered in fish did not cause any significant (p < 0.05) change in the biochemical parameters of sera samples. Nonsignificant changes in the parameters of comet and micronucleus assays were observed between the treated and untreated groups of fishes that further indicated the safety profile of the enterocin ESmr18.

Potential of vermicompost extract in enhancing the biomass and bioactive components along with mitigation of Meloidogyne incognita-induced stress in tomato.

Increasing inorganic fertilizer and pesticide use has been linked to increased health risks for humans and cattle, as well as substantial water and soil contamination. In recent years, vermicomposting has shown to be a viable alternative to chemical pesticides. Vermicompost and vermicompost products such as extract and leachate assist plants in a number of ways. According to recent studies, vermicompost extract (VCE), when used as a supplement, is thought to work as a growth and stress tolerance booster for plants. These liquid supplements also help to suppress a range of pests, such as root knot nematodes. In the present study, neem- and cattle dung-based vermicompost extracts of different concentrations (0, 20, 40, 60, 80 and 100%) were prepared and used for their application against nematode infection in tomato seedlings under laboratory conditions. Apart from its antagonistic action against Meloidogyne incognita, the influence of VCE on plant growth was investigated by analyzing its morphological characteristics in tomato seedlings infected and uninfected with M. incognita. Seeds were pre-soaked in VCE for the seed priming process before being allowed for germination. After 10 days of nematode inoculation, biochemical parameters like protein content, activity of antioxidative enzymes, non-enzymatic antioxidants, stress indices, photosynthetic pigments, proline content and secondary metabolites were also analyzed. The results revealed that neem-based VCE was fatal to second-stage juveniles, with an 82% mortality rate following exposure to the highest dose. When eggs were exposed to 100% VCE, 33.8% of hatching was suppressed, indicating that VCE had an antagonistic effect on nematode egg hatching. Further, all the morphological and biochemical parameters were significantly enhanced in VCE-treated tomato seedlings as compared to untreated seedlings. Stress indices were also found to be significantly lowered by the VCE treatments in the infected plants. The effect of VCE on seedling growth and physiology was shown to be concentration dependent. As a result, the current findings show that VCE has the potential to be used as a plant growth accelerator as well as an environmentally friendly biocontrol agent against nematode pathogenesis in tomato plants.

Combined effects of vermicompost and vermicompost leachate on the early growth of Meloidogyne incognita stressed Withania somnifera (L.) Dunal.

Roots of Withania somnifera, an important medicinal herb, are prone to the infection of Meloidogyne incognita (a root parasitic nematode). The infection lowers the quality and quantity of plant material and poses a challenge in crop cultivation and obtaining desirable yield. In the present study, in vitro inhibitory activity of vermicompost leachate (Vcom-L) was assessed against % hatch and survival of M. incognita in a 96 h assay. Then, Vcom-L was used as soil supplement in combination with vermicompost (Vcom) to evaluate their nematode inhibitory and stress alleviating effect in W. somnifera, in a pot experiment. Root galling intensity and growth performance of nematode-stressed W. somnifera raised from seeds pre-soaked in distilled water (DW), Vcom-L, vermicompost tea (Vcom-T) and different dilutions of Vcom-L were assessed. We observed 79% suppression of egg hatching and 89% juvenile (J2) mortality after 96 h compared to control, at 100% concentration of Vcom-L. Significant reduction in gall formation with increase in growth parameters of seedlings was observed after combined application of Vcom (60% or 100%) + Vcom-L and was evident as enhancement in seedling biomass and contents of chlorophyll and protein. However, proline, total phenolics and malondialdehyde (MDA) content declined significantly in these combinations compared to the control (0% Vcom). Activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidise (APX), guaiacol peroxidise (GPX) and glutathione reductase (GR) declined with Vcom as well as Vcom + Vcom-L and corresponded with decline in the accumulation of reactive oxygen species in leaves. Further, 1:5 and 1:10 dilutions of Vcom-L in combination with Vcom (60%) proved beneficial in mitigating the nematode-induced stress in W. somnifera. Present results showed the potential of Vcom and Vcom-L in standardised combination as an effective strategy in controlling the pathogenicity of M. incognita in medicinal plants such as W. somnifera.

Biomolecular alterations in the early life stages of four food fish following acute exposure of Triclosan.

We investigated the effect of acute concentrations of triclosan (TCS; 96 h exposure and 10d post exposure) on the free amino acid, primary (SDS-PAGE) and secondary (FT-IR) structure of proteins in the embryos/larvae of Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala. A concentration dependent increase in free amino acids, upregulation of polypeptides (100 and 70 kDa in C. carpio, C. idella and L. rohita, 55, 45, 36 kda in C. idella and L. rohita and 22 kDa in all the fish) and a decline in percent area of all the selected peaks of the FT-IR spectra was observed after exposure and recovery period. The decline in percent area was greatest for L. rohita at peak 1080 - 1088 cm-1 (-75.99%) after exposure and at peak 2854 - 2855 cm-1 (-53.59%) after recovery. Curve fitting analysis revealed a decrease in α-helices and increase in ß-sheets in all fish after exposure and recovery period. The results suggest that TCS elicits alterations in biomolecules of fish embryos.

Environmentally relevant concentrations of Triclosan cause transcriptomic and biomolecular alterations in the hatchlings of Labeo rohita.

Suppression (p ≤ 0.05) of antioxidative/detoxification (except GPx and CYP3a) and cytoskeletal (except DHPR) genes but induction of metabolic (except for AST and TRY) and heat shock (except HSP60) genes of Labeo rohita hatchlings after 14 days of exposure to environmentally relevant concentrations of Triclosan (0.0063, 0.0126, 0.0252 and 0.06 mg/L) was followed by an increase (p ≤ 0.05) for most of the genes after 10 days recovery period. After recovery, LDH, ALT, CK, CHY, PA, HSP47 and DHPR declined, while SOD, CAT, GST, GR, GPx, CYP1a, CYP3a, AST, AChE, TRY, HSP60, HSP70, HSc71, HSP90 MLP-3, α-tropomyosin, desmin b and lamin b1 increased over exposure. Peak area of biomolecules (except 3290-3296, 2924-2925 and 2852-2855 cm-1) declined (p ≤ 0.01) more after recovery [except for an increase (p ≤ 0.01) at 1398-1401 cm-1]. CYP3a, CK, HSP90, MLP-3 and secondary structure of amide A are the most sensitive markers for the environmentally relevant concentrations of Triclosan.

Triclosan elicited biochemical and transcriptomic alterations in Labeo rohita larvae.

In the current study, Triclosan (TCS, a commonly used antimicrobial agent) induced alterations in biochemical parameters and gene expression were recorded in the larvae of Labeo rohita after 96 h exposure and 10 days recovery period to find out health status biomarkers. 96 h exposure to 0.06, 0.067 and 0.097 mg/L TCS significantly declined the levels of glucose, triglycerides, urea and uric acid and activity of alkaline phosphatase (ALP), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). There was a non-significant decline in the levels of cholesterol and total protein but albumin and total bilirubin showed no change. After 10 days of recovery period, trend was opposite for glucose, urea and ALP only. Decline in the expression of trypsin and pancreatic amylase and elevation in creatine kinase during exposure to TCS showed a reverse trend after recovery period. However, concentration dependent elevation of chymotrypsin persisted till the end of recovery period. Principal Component Analysis (PCA) showed association of total protein, ALP, GOT, creatine kinase and pancreatic amylase with PC1 after exposure as well as recovery period. Therefore, these can be considered as important biomolecules for identification of health status of TCS stressed fish.

Genomic markers for the biological responses of Triclosan stressed hatchlings of Labeo rohita.

Triclosan (TCS) used commonly in pharmaceuticals and personal care products has become the most common pollutant in water. Three-day-old hatchlings of an indigenous fish, Labeo rohita, were given 96h exposure to a nonlethal (60 µg L-1) and two moderately lethal concentrations (67 and 97 µg L-1) of TCS and kept for 10 days of recovery for recording transcriptomic alterations in antioxidant/detoxification (SOD, GST, CAT, GPx, GR, CYP1a and CYP3a), metabolic (LDH, ALT and AST) and neurological (AchE) genes and DNA damage. The data were subjected to principal component analysis (PCA) for obtaining biomarkers for the toxicity of TCS. Hatchlings were highly sensitive to TCS (96h LC50 = 126 µg L-1 and risk quotient = 40.95), 96h exposure caused significant induction of CYP3a, AChE and ALT but suppression of all other genes. However, expression of all the genes increased significantly (except for a significant decline in ALT) after recovery. Concentration-dependent increase was also observed in DNA damage [Tail Length (TL), Tail Moment (TM), Olive Tail Moment (OTM) and Percent Tail DNA (TDNA)] after 96 h. The damage declined significantly over 96h values at 60 and 67 µg L-1 after recovery, but was still several times more than control. TCS elicited genomic alterations resulted in 5-11% mortality of exposed hatchlings during the recovery period. It is evident that hatchlings of L. rohita are a potential model and PCA shows that OTM, TL, TM, TDNA, SOD and GR (association with PC1 during exposure and recovery) are the biomarkers for the toxicity of TCS. Graphical abstract.