KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function 1 . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) 2 , the most common embryonal brain tumor in children, and pineoblastoma 3 . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST 4 . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

Computational exploration of novel antimicrobial modalities targeting fucose-binding lectins and ribosomes in Mycobacterium smegmatis using tRNA-encoded peptides.

tRNA-Encoded Peptides (tREPs), encoded by small open reading frames (smORFs) within tRNA genes, have recently emerged as a new class of functional peptides exhibiting antiparasitic activity. The discovery of tREPs has led to a re-evaluation of the role of tRNAs in biology and has expanded our understanding of the genetic code. This presents an immense, unexplored potential in the realm of tRNA-peptide interactions, paving the way for groundbreaking discoveries and innovative applications in various biological functions. This study explores the antimicrobial potential of tREPs against protein targets by employing a computational method that uses verified data sources and highly recognized predictive algorithms to provide a sorted list of likely antimicrobial peptides, which were then filtered for toxicity, cell permeability, allergenicity and half-life. These peptides were then docked with screened protein targets and computationally validated using molecular dynamics (MD) simulations for 150 ns and the binding free energy was estimated. The peptides Pep2 (VVLWRKPRVRKTG) and Pep6 (HRLRLRRRKPWW) exhibited good binding affinities of -110.5 +/- 2.5 and -129.0 +/- 3.9, respectively, with RMSD values of 0.4 and 0.25 nm against the fucose-binding lectin (7NEF) and the 30S ribosome of Mycobacterium smegmatis (5O5J) protein targets. The 7NEF-Pep2 and 5O5J-Pep6 complexes indicated higher negative binding free energies of -52.55 kcal/mol and -55.52 kcal/mol respectively, as calculated by Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). Thus, the tREPs derived peptides designed as a part of this study, provide novel approaches for potential anti-bacterial therapeutic modalities.Communicated by Ramaswamy H. Sarma.

Clofarabine monotherapy in aggressive, relapsed and refractory Langerhans cell histiocytosis.

Over 50% of patients with systemic LCH are not cured with front-line therapies, and data to guide salvage options are limited. We describe 58 patients with LCH who were treated with clofarabine. Clofarabine monotherapy was active against LCH in this cohort, including heavily pretreated patients with a systemic objective response rate of 92.6%, higher in children (93.8%) than adults (83.3%). BRAFV600E+ variant allele frequency in peripheral blood is correlated with clinical responses. Prospective multicentre trials are warranted to determine optimal dosing, long-term efficacy, late toxicities, relative cost and patient-reported outcomes of clofarabine compared to alternative LCH salvage therapy strategies.

Computational Prediction of Potential Vaccine Candidates From tRNA Encoded peptides (tREP) Using a Bioinformatic Workflow and Molecular Dynamics Validations.

Transfer RNAs (tRNA) are non-coding RNAs. Encouraged by biological applications discovered for peptides derived from other non-coding genomic regions, we explore the possibility of deriving epitope-based vaccines from tRNA encoded peptides (tREP) in this study. Epitope-based vaccines have been identified as an effective strategy to mitigate safety and specificity concerns observed in vaccine development. In this study, we explore the potential of tREP as a source for epitope-based vaccines for virus pathogens. We present a computational workflow that uses verified data sources and community-validated predictive tools to produce a ranked list of plausible epitope-based vaccines starting from tRNA sequences. The top epitope, bound to the predicted HLA molecule, for the virus pathogen is computationally validated through 200 ns molecular dynamics (MD) simulations followed by binding free energy calculations. The simulation results indicate that two tRNA encoded epitope-based vaccines, RRHIDIVV and IMVRFSAE for Mamastrovirus 3 and Norovirus GII, respectively, are likely candidates. Peptides originating from tRNAs provide unexplored opportunities for vaccine design. Encouraged by our previous experimental study, which established the inhibitory properties of tREPs against infectious parasites, we have proposed a computationally validated set of peptides derived from tREPs as vaccines for viral pathogens.

In silico investigations and molecular insights for designing tRNA-encoded peptides as potential therapeutics for targeting over-expressed receptors in breast cancer.

tRNA- Encoded Peptides (tREPs) have recently been discovered as new functional peptides and hold promise as therapeutics for anti-parasitic applications. In this study, in silico investigations were conducted to design tRNA-encoded peptides with the potential to target over-expressed receptors in breast cancer cells. tRNA genes were translated into corresponding peptides (tREPs) using computational tools. The tREPs, which were predicted as anticancer peptides, were then screened for various ADMET properties. Molecular docking studies were conducted for three cancer target receptors, the Estrogen Receptor (ER), Peroxisome Proliferator-Activated Receptor (PPAR) and the Epidermal Growth Factor Receptor (EGFR). Based on the docking results, specific tREPs were screened and molecular dynamics simulations were performed, and the binding energies were further explored using MMPBSA calculations. The peptide Pep1 (DWIAWRHHNDIVSWLTCGPRFKSWS) and Pep2 (GFIAWWSRHLELAQTRFKSWWS) exhibited a good binding affinity against the Estrogen Receptor (ER) and the Peroxisome Proliferator-Activated Receptor Alpha (PPAR) cancer target. The Pep1-ER and Pep1-PPAR complex maintained an average of two hydrogen bonds throughout the simulation and demonstrated a higher negative binding free energy of -72.27 kcal/mol and -65.16 kcal/mol respectively, as calculated by MMPBSA. Therefore, the tREPs designed as anticancer peptides in this study provide novel approaches for potential anticancer therapeutic modalities.Communicated by Ramaswamy H. Sarma.

Prevalence of goitre and iodine deficiency among school children (6-12 years) in rural areas of North Karnataka, India: A cross-sectional survey, 2016-19.

Introduction: Iodine deficiency disorders (IDD) have remained an unresolved public health problem in India. In this survey, we have estimated the prevalence of IDD among 6-12 years of school children in rural areas of north Karnataka, India and estimated the prevalence of low iodine content (<15 ppm) in salt at the household level and urine iodine excretion in this population. Material and Methods: In this cross-sectional survey, we recruited 16,827 children between 6 and 12 years of age through multistage sampling from six districts. Goitre was examined clinically for all children. Household-level salt iodine estimation and urinary iodine estimation were carried out among a subset of the participants. Results: Overall prevalence of goitre was 17.1% (95% CI: 16.5, 17.7). Out of this, 76.7% (n = 2116) had Grade-1 goitre, and 23.7% (n = 656) had Grade-2 goitre. The prevalence of goitre was higher among females (17.9%, vs. male 16.4%, P < 0.05). The prevalence of low iodine content (<15 ppm) in salt was 48.5% (95%CI: 46.7, 50.3). The overall median iodine excretion in urine was 85 µg/L (IQR: 60-150 µg/L). In total, 37.2% (n = 601) had mild iodine deficiency, 5.2% (n = 84) had moderate deficiency, and 10.1% (n = 163) had severe deficiency. All parameters showed high inter- and intradistrict variations. Conclusion: North Karnataka has a high goitre prevalence. Low use of iodized salt can be a major reason for the high prevalence of the condition. Ensuring the availability of iodized salt in this region and periodic surveillance to measure the impact of the programme should be the priority in this region.

Determination of 73 multi-class pesticides in okra (Abelmoschus esculentus L.) fruits using LC-MS/MS and GC-MS/MS and estimation of analytical uncertainty of measurement.

This study developed a method to simultaneously determine 73 multi-class pesticides in okra fruit using LC-MS/MS and GC-MS/MS. The sample was extracted with acetonitrile and subsequent clean-up through dispersive-SPE method. The quantification level of the technique was 0.01 µg g-1 and compliance to the MRLs fixed by the regulatory bodies like EU and FSSAI. The recovery at 10, 50, and 100 µg kg-1 spiked levels; intra and inter-day precision at 50 µg kg-1 were found within 70-120% with RSD less than 15% with LC-MS/MS and GC-MS/MS. Measurement uncertainty was in the range of 1.81 to 12.91 µg kg-1 estimated at 50 µg kg-1. The matrix effects were slightly higher for LC than GC-compatible pesticides. Risk assessment for pesticides detected in the field and market samples found no hazardous to the consumers except profenofos. The proposed method is highly sensitive, reproducible for the complex matrix like okra, and meets the regulatory standards.

Determination of chlorantraniliprole 18.5% SC in the paddy ecosystem and its risk assessment.

Chlorantraniliprole belongsto theanthranilic diamide group is widely used against broad range of lepidopteron pests in a variety of vegetable and rice pests includingyellow rice stem borer and leaf folder. Supervised field trials were conducted duringRabi (2018-2019) and Kharif (2019) to evaluate the dissipation pattern and risk assessment of chlorantraniliprole 18.5% SC in paddy ecosystem following foliar application at 30 and 60 g a.i. ha-1 in two different cropping seasons.Modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique was used for the extraction of CAP residues with acetonitrile and determined by LC-MS/MS (ESI +).The limit of quantification (LOQ) was 0.01 µg g-1 for paddy leaf, straw, husk, and brown rice, respectively and 0.005 µg g-1 for soil. The average recoveries obtained were 84.30-88.92% from paddy leaf, 94.25-97.81% from straw, 90.21-93.38% from husk, 93.57-96.40% from brown rice and 89.93-91.14% from soil. The residues in paddy leaf dissipated within 35-40 days with a half-life of 4.33-5.07 days in Rabi and 3.92-4.86 days in Kharif at 30 and 60 g a.i. ha-1, respectively. The residues in soil dissipated within 15-21 days with a half-life of 14.44-15.75 days in Rabi and 13.33-14.44 days in Kharif at respective doses. At harvest chlorantraniliprole residues were not detected in straw, husk, and brown rice. The dietary risk of paddy leaf (green fodder) for cattle was found safe for consumption as the hazard index is less than one. Soil ecological risk assessment was found to be less than one (RQ < 0.1) for earthworms (Eisenia foetida) and arthropods (Aphidiusrhopalosiphi). The presentmethod could be useful inthe analysis ofchlorantraniliproleresidues in different cereals and vegetable crop ecosystems and application at recommended dose is safe for the final produce at harvest.

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework.

In silico based multi-epitope vaccine design against norovirus.

Norovirus (NoV) belongs to the Calciviridae family that causes diarrhoea, vomiting, and stomach pain in people who have acute gastroenteritis (AGE). Identifying multi-epitope dependent vaccines for single stranded positive sense viruses such as NoV has been a long due. Although efforts have been in place to look into the candidate epitopes, understanding molecular mimicry and finding new epitopes for inducing immune responses against the T/B-cells which play an important role for the cell-mediated and humoral immunity was not dealt with in great detail. The current study focuses on identifying new epitopes from various databases that were filtered for antigenicity, allergenicity, and toxicity. The adjuvant ß-defensin along with different linkers were used for vaccine construction. Further, the binding relationship between the vaccine construct and toll-like immune receptor (TLR3) complex was determined using a molecular docking analysis, followed by molecular dynamics simulation of 100 ns. The vaccine candidate developed expresses good solubility with a score of 0.530, Z-score of -4.39 and molecular docking score of -140.4 ± 12.1. The MD trajectories reveal that there is a stability between TLR3 and the developed vaccine candidate with an average of 0.91 nm RMSD value and also the system highest occupancy H-bond formed between GLU127 of TLR3 and TYR10 of vaccine candidate (61.55%). Four more H-bonds exist with an occupancy of more than 32% between TLR3 and the vaccine candidates which makes it stable. Thus, the multi-epitope based vaccine developed in the present study forms the basis for further experimental investigations to develop a potentially good vaccine against NoV.Communicated by Ramaswamy H. Sarma.

A Descriptive Analysis of Unique Disability Identification Card (UDID)-Certified Visually Disabled Patients at a Tertiary Eye Care Center in Central India.

Objective In this study, we aimed to examine the demographic characteristics, causes, and severity of visual disability and the reasons for seeking disability certificates among Unique Disability Identification Card (UDID)-certified visually disabled patients at a tertiary eye care center in central India. Materials and methods A retrospective observational analysis of medical records and data from the UDID portal involving 600 visually disabled individuals who were certified between February 2019 to March 2022 was performed. Demographic characteristics, diagnosis of the ocular disease, primary etiology, and percentage and grade of visual disability, as well as the main reasons for seeking a visual disability certificate, were analyzed statistically. Best-corrected visual acuity of less than 6/24 to 3/60 or a visual field less than 40 degrees to 10 degrees around the center of fixation or hemianopia involving the macula in the better eye were included in the low-vision category. Best corrected visual acuity of less than 3/60 to "no light perception" or visual field less than 10 degrees around the center of fixation in the better eye were included in the blindness category. Results Out of the total 600 patients, 214 (35.67%) were in the age group of 11-30 years. There were more males (63.67%) than females (36.33%) in the study. Four hundred patients (66.67%) had 100% disability. Retinal diseases (n=229, 48.50%) including retinitis pigmentosa (RP) (n=130, 21.67%) were the most common cause of visual disability. Travel concessions and Government allowances were the most common reasons for seeking a disability certificate. Conclusion The study highlights the leading causes of visual disability, and RP was found to be the most common one. Avoidance of consanguineous marriages and genetic counseling should be made mandatory to prevent blindness due to RP. We recommend the widespread institution of eye care facilities, increasing the availability of healthcare facilities to all, and community education to eliminate avoidable blindness. This study provides key data to the Government to implement new policies or change the existing ones, plan for future strategies, and prioritize the rehabilitation of visually disabled individuals. Government programs to increase awareness among unregistered visually disabled patients regarding the benefits and rehabilitative measures like UDID card and low vision aids is the need of the hour.

ProAll-D: protein allergen detection using long short term memory - a deep learning approach.

Background: An allergic reaction is the immune system's overreacting to a previously encountered, typically benign molecule, frequently a protein. Allergy reactions can result in rashes, itching, mucous membrane swelling, asthma, coughing, and other bizarre symptoms. To anticipate allergies, a wide range of principles and methods have been applied in bioinformatics. The sequence similarity approach's positive predictive value is very low and ineffective for methods based on FAO/WHO criteria, making it difficult to predict possible allergens. Method: This work advocated the use of a deep learning model LSTM (Long Short-Term Memory) to overcome the limitations of traditional approaches and machine learning lower performance models in predicting the allergenicity of dietary proteins. A total of 2,427 allergens and 2,427 non-allergens, from a variety of sources, including the Central Science Laboratory and the NCBI are used. The data was divided 80:20 for training and testing purposes. These techniques have all been implemented in Python. To describe the protein sequences of allergens and non-allergens, five E-descriptors were used. E1 (hydrophilic character of peptides), E2 (length), E3(propensity to form helices), E4(abundance and dispersion), and E5 (propensity of beta strands) are used to make the variable-length protein sequence to uniform length using ACC transformation. A total of eight machine learning techniques have been taken into consideration. Results: The Gaussian Naive Bayes as accuracy of 64.14 %, Radius Neighbour's Classifier with 49.2 %, Bagging Classifier was 85.8 %, ADA Boost was 76.9 %, Linear Discriminant Analysis has 76.13 %, Quadratic Discriminant Analysis was 84.2 %, Extra Tree Classifier was 90%, and LSTM is 91.5 %. Conclusion: As the LSTM, has an AUC value of 91.5 % is regarded best in predicting allergens. A web server called ProAll-D has been created that successfully identifies novel allergens using the LSTM approach. Users can use the link https://doi.org/10.17632/tjmt97xpjf.1 to access the ProAll-D server and data.

Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders.

Targeted protein degradation (TPD) holds immense promise for drug discovery, but mechanisms of acquired resistance to degraders remain to be fully identified. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation, yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are nonessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation.

Activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase pathway in childhood B-cell acute lymphoblastic leukemia.

RAS mutations are frequently observed in childhood B-cell acute lymphoblastic leukemia (B-ALL) and previous studies have yielded conflicting results as to whether they are associated with a poor outcome. We and others have demonstrated that the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK) pathway can be activated through epigenetic mechanisms in the absence of RAS pathway mutations. Herein, we examined whether MAPK activation, as determined by measuring phosphorylated extracellular signal-regulated kinase (pERK) levels in 80 diagnostic patient samples using phosphoflow cytometry, could be used as a prognostic biomarker for pediatric B-ALL. The mean fluorescence intensity of pERK (MFI) was measured at baseline and after exogenous stimulation with or without pretreatment with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Activation levels (MFI stimulated/MFI baseline) ranged from 0.76 to 4.40 (median = 1.26), and inhibition indexes (MFI stimulated/MFI trametinib stimulated) ranged from 0.439 to 5.640 (median = 1.30), with no significant difference between patients with wildtype versus mutant RAS for either. Logistic regression demonstrated that neither MAPK activation levels nor RAS mutation status at diagnosis alone or in combination was prognostic of outcome. However, 35% of RAS wildtype samples showed MAPK inhibition indexes greater than the median, thus raising the possibility that therapeutic strategies to inhibit MAPK activation may not be restricted to patients whose blasts display Ras pathway defects.

Acute myeloid leukemia with an MN1-ETV6 fusion in a young child with Down syndrome.

Myeloid leukemia of Down syndrome (ML-DS) in young children is associated with distinct clinical and biological features and is typically initiated with oncogenic mutations in the X-linked megakaryocytic transcription factor GATA1. Here we present a 3-yr-old child with DS diagnosed with acute myeloid leukemia (AML), which lacks typical immunophenotypic and molecular characteristics of ML-DS, including GATA1 mutations. The leukemic blasts were found to have an MN1-ETV6 gene fusion, a high-risk oncofusion not previously described in DS patients. This report highlights the importance of immunophenotypic, cytogenetic, and molecular characterization of ML-DS for identification of rare cases with unique features that may benefit from treatment protocols that are more intensive than those developed for patients with typical GATA1 mutant ML-DS.

Simultaneous determination of pesticide residues in pomegranate whole fruit and arils using LC-MS/MS.

An analytical method in pomegranate whole fruits and arils was developed in LC-MS/MS and validated as per SANTE/12682/2019. Samples were extracted following acetonitrile-based modified QuEChERS protocol. The method was linear and the coefficient of determination ranged between 0.998 and 0.999. Through this method, all the pesticides were detected and quantified at 10 µg kg-1. The accuracy test at 10, 50, and 100 µg kg-1 spiking level recorded recovery between 70 and 120% and RSD less than 15% in both matrices. No significant matrix effect was observed for most pesticides. Intra (RSDr) and inter-day (RSDwr) precision estimated at 50 µg kg-1 found acceptable RSD in both matrices. Measurement uncertainty at 50 µg kg-1 was in the range of 4.02 to 16.12 µg kg-1. Quantifying pesticides in pomegranate whole fruits, peel, and arils using the proposed method is highly suitable and reproducible for 74 pesticides in a short run time of 25.00 min.

Production, stability and degradation of Trichoderma gliotoxin in growth medium, irrigation water and agricultural soil.

Gliotoxin produced by Trichoderma virens is inhibitory against various phytopathogenic fungi and bacteria. However, its stability in soil-ecosystem has not yet been well-defined. This study aimed to decipher its persistence and behaviour in growth media, irrigation water and soil ecosystems. Gliotoxin production was noticed at logarithmic growth phase and converted into bis-thiomethyl gliotoxin at late stationary growth phase of T. virens in acidic growth medium. But, no gliotoxin production was observed in neutral and alkaline growth medium. Gliotoxin was stable for several days in acidic water but degraded in alkaline water. Degradation of gliotoxin was more in unsterile soil than sterile soil and also that was higher under wet soil than dry soil. Degradation of gliotoxin was hastened by alkaline pH in wet soil but not in dry soil. Under unsterile soil conditions, high soil moisture increased the degradation of gliotoxin and the degradation of gliotoxin occurred quickly in alkaline soil (in 5 days) compared to acidic soil (in 10 days). Under sterile soil conditions, high soil moisture also enhanced the degradation of gliotoxin but level of degradation was less compared to unsterile conditions. Thus, gliotoxin stability is influenced mainly by the soil wetness, soil microbial community and pH conditions.

Simultaneous determination, dissipation and decontamination of fungicides applied on cabbage using LC-MS/MS.

A method for simultaneous determination of carbendazim and tebuconazole residues in cabbage was developed and validated in LC-MS/MS. Samples were extracted and purified following the modified QuEChERS procedure, which enabled the elution of carbendazim and tebuconazole at 0.96 and 5.31 min, respectively. LOD and LOQ were 0.0005 and 0.0015 mg kg-1, respectively. Mean recovery was in the range of 78.94 to 104.89% for carbendazim and 76.07 to 98.62% for tebuconazole. The field samples recorded residues of 0.274 and 0.481 mg kg-1; and 0.194 and 0.392 mg kg-1 at single and double dose for carbendazim and tebuconazole, respectively. Half-life values were 2.17 and 2.99 for carbendazim and 2.74 and 2.81 for tebuconazole at single and double dose, respectively. Decontamination with saltwater wash followed by cooking and lemon water wash found superior in the removal of residues more than 90%.