Elucidation of ejaculatory bulb proteins in Bemisia tabaci Asia-1 and Asia II-1 and confirmation of their mating transfer via RNAi.

BACKGROUND: Bemisia tabaci, a significant agricultural pest in Asia, contains distinct genetic groups, Asia-1 and Asia II-1. Understanding its reproductive biology, particularly the role of ejaculatory bulb proteins (EBPs) in mating, is crucial. However, EBPs in B. tabaci were not well characterised until this study. METHODS AND RESULTS: The EBPs have been characterised in the Asia-1 and Asia II-1 genetic groups of the whitefly B. tabaci, prevalent in Asia. The transcriptomic analysis yielded over 40,000,000 and 30,000,000 annotated transcripts, respectively, from Asia II-1 and Asia-1. Differential gene expression revealed the presence of 270 upregulated and 198 downregulated genes, with significant differences between these two genetic groups. Orphan genes (1992 numbers) were identified in both genetic groups. We report, for the first time, full-length sequences of EBP genes from B. tabaci. The 10 EBPs each deduced in B. tabaci Asia-1 and Asia II-1 are structurally akin to chemosensory proteins having four conserved cysteine residues. Additionally, we did domain analysis, protein structure prediction, mapping of these EBPs in the chromosomes of B. tabaci, and phylogenetic analysis to track their evolutionary lineage. We have specifically demonstrated the transfer of EBPs from males to females during mating using qPCR and further validated the transfer of EBPs through RNAi. Specifically, we targeted the highly expressed EBPs (EBP-3, 7, and 8 in BtAsia1; EBP-8, 9, and 10 in BtAsia II-1) through feeding bioassays of dsRNAs. Tracking by qPCR revealed that the females, when mated with dsRNA-treated males, did not show expression of the specific EBP, suggesting that the silencing of these genes in males hinders the transfer of EBP to females during mating. CONCLUSION: Our findings provide novel insights into the genomic contours of EBPs in B. tabaci and underscore the potential of RNAi-based strategies for pest management by disrupting the reproductive processes.

Metabolomics-Driven Mining of Metabolite Resources: Applications and Prospects for Improving Vegetable Crops.

Vegetable crops possess a prominent nutri-metabolite pool that not only contributes to the crop performance in the fields, but also offers nutritional security for humans. In the pursuit of identifying, quantifying and functionally characterizing the cellular metabolome pool, biomolecule separation technologies, data acquisition platforms, chemical libraries, bioinformatics tools, databases and visualization techniques have come to play significant role. High-throughput metabolomics unravels structurally diverse nutrition-rich metabolites and their entangled interactions in vegetable plants. It has helped to link identified phytometabolites with unique phenotypic traits, nutri-functional characters, defense mechanisms and crop productivity. In this study, we explore mining diverse metabolites, localizing cellular metabolic pathways, classifying functional biomolecules and establishing linkages between metabolic fluxes and genomic regulations, using comprehensive metabolomics deciphers of the plant's performance in the environment. We discuss exemplary reports covering the implications of metabolomics, addressing metabolic changes in vegetable plants during crop domestication, stage-dependent growth, fruit development, nutri-metabolic capabilities, climatic impacts, plant-microbe-pest interactions and anthropogenic activities. Efforts leading to identify biomarker metabolites, candidate proteins and the genes responsible for plant health, defense mechanisms and nutri-rich crop produce are documented. With the insights on metabolite-QTL (mQTL) driven genetic architecture, molecular breeding in vegetable crops can be revolutionized for developing better nutritional capabilities, improved tolerance against diseases/pests and enhanced climate resilience in plants.

Estimation of Free Phenytoin Concentration in Critically Ill Patients with Hypoalbuminemia: Direct-measurement vs Traditional Equations.

Background: In critically ill patients with low albumin, dose individualization of phenytoin is a challenge. The currently used Sheiner-Tozer equation does not accurately predict the free phenytoin concentration in serum and can result in incorrect dose modifications. The best measure to advocate in these patients is the direct-measurement of free phenytoin concentration. Aims and objectives: Phenytoin exhibits complex pharmacokinetics, requiring careful therapeutic drug monitoring. This study aimed to compare the accuracy of the established Sheiner-Tozer calculation method against the direct-measurement of free phenytoin concentration in serum by high performance liquid chromatography in critically ill patients with low albumin. Materials and methods: Blood specimens for direct-measurement of both total and free phenytoin concentration were obtained from 57 patients with hypoalbuminemia monitored in the intensive care unit. Results: The median [inter-quartile range (IQR)] for Sheiner-Tozer equation calculated total phenytoin concentration and direct-measured total was 17.14 (10.63-24.53) and 9.82 (6.02-13.85) µg mL-1, respectively. Approximately 53 and 5% of patients were found to be subtherapeutic and supratherapeutic for direct-measured total phenytoin concentrations, respectively. In contrast, on applying the Sheiner-Tozer calculation, 23 and 40% had subtherapeutic and supratherapeutic concentrations, respectively, for total phenytoin concentration. The median (IQR) for direct-measured, routine and Sheiner-Tozer equation calculated free phenytoin concentration were 1.92 (1.06-2.76), 0.98 (0.60-1.39), and 1.71 (1.06-2.45) µg mL-1, respectively. Only 45.7% of patients were in agreement with respect to the therapeutic category when direct-measured free was compared against routine calculation free. Conclusion: In patients with low albumin, free phenytoin concentration based on the Sheiner-Tozer corrected equation accurately classified patients based on their therapeutic category of free phenytoin in 73.7% of patients. Hence, for individualization of phenytoin dosage in critically ill patients with low albumin, we recommend direct-measurement of free phenytoin concentration. How to cite this article: Wilfred PM, Mathew S, Chacko B, Prabha R, Mathew BS. Estimation of Free Phenytoin Concentration in Critically Ill Patients with Hypoalbuminemia: Direct-measurement vs Traditional Equations. Indian J Crit Care Med 2022;26(6):682-687.

Systemic exposure to 5-fluorouracil and its metabolite, 5,6-dihydrofluorouracil, and development of a limited sampling strategy for therapeutic drug management of 5-fluorouracil in patients with gastrointestinal malignancy.

AIMS: 5-Fluorouracil (5-FU) is widely used in combination chemotherapy, and literature suggests pharmacokinetic-guided dosing to improve clinical efficacy and reduce toxicity. This study aimed to determine the pharmacokinetic exposure of both 5-FU and its metabolite, 5,6-dihydrofluorouracil (DHFU), in patients with gastrointestinal malignancy and to establish a simplified strategy to assist in therapeutic drug management for dose optimization. METHODS: This was a prospective, observational study, performed in 27 patients diagnosed with gastrointestinal malignancy who were prescribed 5-FU. Multiple samples were collected per patient over the slow bolus (15-20 min) and continuous infusion period (over 44 h) in doses 1 and 3, and the concentrations of 5-FU and DHFU were measured. RESULTS: A higher proportion of patients had exposures within the therapeutic range in dose 3 (50%) as compared to dose 1 (37.5%) with 5-FU. There was an association between delayed time to maximum concentration of DHFU and a high maximum concentration of 5-FU. A limited sampling strategy was developed with 4 samples, 2 during the bolus period and 2 during the continuous period (at 18 h and the end of infusion), which accurately predicted the total area under the curve of 5-FU. CONCLUSION: Using body surface area-based dosing with 5-FU, 50-60% of patients were outside of the therapeutic range. In the absence of genotype testing, measurement of the metabolite DHFU could be a phenotypical measure of dihydropyrimidine dehydrogenase enzyme activity. A limited sampling strategy was developed in patients who were prescribed a combination regimen of slow bolus, followed by a 44-hour continuous infusion of 5-FU to assist in the therapeutic drug management of patients.

The effect of phosphodiesterase-5 inhibitor, tadalafil, on in vitro potassium chloride-induced contractions of isolated human ureteral tissue.

INTRODUCTION: Drugs causing ureteral relaxation are used for medical expulsive therapy (MET) for stones. We investigated the in vitro ability of tadalafil to cause relaxation of potassium chloride (KCl)-induced contractions of isolated human ureteral tissue. MATERIALS AND METHODS: Eight grossly normal proximal ureteral tissues were collected from the radical and donor nephrectomy specimen. The standard organ bath protocol was followed. Ureteral contractions were induced with 80 mM KCl before and after exposure to tadalafil. RESULTS: The median amplitude and frequency of KCl-induced contractions and the median area under the contractility curve (AUCC) after exposure to 20 µM tadalafil showed significant reductions compared to that of before exposure to tadalafil (7.87 cm, 3.79/min, and 2.98 cm2, respectively, versus 9.37 cm, 4.48/min, and 4.50 cm2, respectively; P = 0,026, 0.008, and 0.008, respectively). After exposure to 40 µM tadalafil, the median amplitude and frequency of KCl-induced contractions and AUCC (4.50 cm, 2.56/min, and 0.92 cm2, respectively) showed significant reductions compared to that of before exposure to tadalafil (7.62 cm, 3.88/min, and 3.32 cm2, respectively; P = 0.008, 0.016, and 0.008, respectively). However, reductions in the parameters after exposure to 20 µM and 40 µM tadalafil were similar (P = 0.065, 0.195, and 0.130, respectively, for median amplitude, frequency, and AUCC). CONCLUSION: Tadalafil reduces KCl-induced contractions of isolated human ureteral tissue in vitro. No incremental relaxations in contractions occurred by increasing the dose of tadalafil from 20 µM to 40 µM.

A Nonparametric Pharmacokinetic Approach to Determine the Optimal Dosing Regimen for 30-Minute and 3-Hour Meropenem Infusions in Critically Ill Patients.

BACKGROUND: Pharmacokinetics of meropenem differ widely in the critically ill population. It is imperative to maintain meropenem concentrations above the inhibitory concentrations for most of the interdose interval. A population pharmacokinetic/pharmacodynamic model was developed to determine the probability of target attainment for 3-hour and 30-minute infusion regimens in this population. METHODS: This study was performed in an intensive care setting among adult patients who were initiated on meropenem at a dose of 1000 mg. Multiple blood specimens were collected at predetermined time points during the interdose period, and meropenem concentrations were measured using high performance liquid chromatography. Using Pmetrics, a pharmacokinetic/pharmacodynamic model was developed and validated. Monte Carlo simulation was performed, and probability of target attainment (100% T > minimum inhibitory concentration (MIC), with a probability >0.9) for doubling MICs was determined for different regimens of meropenem. RESULTS: A 2-compartment multiplicative gamma error model best described the population parameters from 34 patients. The pharmacokinetic parameters used in the final model were Ke (elimination rate constant from the central compartment), Vc (volume of distribution of central compartment), KCP and KPC (intercompartmental rate constants), and IC2 (the fitted amount of meropenem in the peripheral compartment). Inclusion of creatinine clearance (CLcreat) and body weight as covariates improved the model prediction (Ke = Ke0 × (Equation is included in full-text article.), Vc = Vc0 × Weight). The Ke and Vc [geometric mean (range)] of the individuals were 0.54 (0.01-2.61)/h and 9.36 (4.35-21.62) L, respectively. The probability of attaining the target, T > MIC of 100%, was higher for 3-hour infusion regimens compared with 30-minute infusion regimens for all ranges of CLcreat. CONCLUSIONS: This study emphasizes that extended regimens of meropenem are preferable for treating infections caused by bacteria with higher MICs. The nonparametric analysis using body weight and CLcreat as covariate adequately predicted the pharmacokinetics of meropenem in critically ill patients with a wide range of renal function.

Therapeutic Drug Monitoring of Levetiracetam and Lamotrigine: Is There a Need?

BACKGROUND: This study was a retrospective assessment of the therapeutic drug monitoring data collected for levetiracetam and lamotrigine from a clinical setting. The proportion of patients in relation to the therapeutic ranges for serum concentrations of lamotrigine and levetiracetam was estimated, and the influence of age and anticonvulsant comedications on their clearances were studied. METHODS: Information on levetiracetam (2011-2013) and lamotrigine (2008-2013) dose, trough concentration, age, sex, body weight, and anticonvulsant comedications prescribed was obtained from the therapeutic drug monitoring register and archived medical records. Patients were categorized into 4 groups based on anticonvulsant comedications and further divided into 3 subgroups based on age (a: <9 years; b: 9-17 years; c: ≥18 years). In each subgroup, the proportion of patients who achieved trough concentrations in the therapeutic range for levetiracetam and lamotrigine was computed. Apparent clearance (CL/F) was compared across subgroups by 1-way analysis of variance, and factors which significantly predicted CL/F were identified by stepwise multiple linear regression. RESULTS: Overall, 348 (330 patients) and 706 (493 patients) samples for levetiracetam and lamotrigine were included in the analysis. Of these, 56.9% and 72.4% were within, 43.1% and 23.9% below, 0% and 3.7% above the therapeutic range for levetiracetam and lamotrigine, respectively. A significant difference in CL/F was noted across subgroups for levetiracetam (P < 0.001) and lamotrigine (P < 0.001). Age <9 years, age ≥18 years, and inducer comedications significantly predicted CL/F for levetiracetam. For lamotrigine, inhibitor comedications, age <9 years, inducer comedications, and age 9-17 years significantly predicted CL/F. CONCLUSIONS: These findings emphasize the need to monitor relatively newer anticonvulsants, lamotrigine and levetiracetam, especially among children and when other anticonvulsant comedications are prescribed or discontinued in the treatment regimen.

Clinical efficacy and pharmacokinetics of tacrolimus in children with steroid-resistant nephrotic syndrome.

BACKGROUND: Tacrolimus has gained acceptance in the management of steroid-resistant nephrotic syndrome (SRNS) in children. Due to limited data, therapeutic range is extrapolated from pediatric renal transplant recipients. This study was designed to assess therapeutic efficacy of tacrolimus in children with SRNS and its correlation with inter-dose area under concentration curve (AUC0-12 h) and trough concentration (C0). METHODS: Pre dose, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 8, and 12 h after drug administration blood samples were collected in 25 children who were on tacrolimus for a minimum of 3 months and AUC0-12 h was calculated. RESULTS: There was an 80% (20/25) response rate with 64% (16/25) children achieving complete remission. Median C0 in remission was higher than in relapse group (2.95 ng/ml, versus 1.20 ng/ml, p = 0.005). Median AUC0-12 h in remission was higher compared to those in relapse group (79.75 versus 35.15 µg × h/l; p = 0.025). Maximum concentration after drug administration (Cmax) among the groups was not significantly different. There was a significant correlation between C0 and AUC0-12 h (r = 0.79); and Cmax and AUC0-12 h (r = 0.84). Five patients had a rise in serum creatinine, of which four were still proteinuric and had lower C0 and AUC0-12 h. No other adverse effect was noted. CONCLUSIONS: Tacrolimus had beneficial clinical response in SRNS. Target C0 and AUC0-12 h level for treatment remission was higher than those in relapse in children with SRNS but was lower than required in transplant recipient.

Exposure to Mycophenolic Acid and Its Clinical Response in an Indian Pediatric Population with Nephrotic Syndrome.

Background: Children with nephrotic syndrome experience many side effects and frequent relapses when treated with steroids and other drugs. Mycophenolic acid (MPA) is one of the effective and least toxic drug for the treatment of nephrotic syndrome. This drug needs to be monitored for maximal efficacy and minimal toxicity. The therapeutic reference range for this drug is not established for the aforementioned patient population of Indian origin. Materials and Methods: In this observational study, children with nephrotic syndrome on mycophenolate mofetil were followed up for a minimum duration of three months. Following this, their clinical status (relapse/remission) was determined and the mycophenolate exposure was measured for over 12 hours. Results: A total of 34 participants were included, with 17 (50%) in relapse. Median MPA Area under the curve over 12 hours (AUC0-12h) (36.5 µg·h/ml) in the remission group differed significantly compared to that in the relapse group (17.2 µg·h/ml). Conclusion: Higher exposure to MPA AUC0-12h is associated with clinical remission of pediatric nephrotic syndrome.

In silico mutation of aromatic with aliphatic amino acid residues in Clostridium perfringens epsilon toxin (ETX) reduces its binding efficiency to Caprine Myelin and lymphocyte (MAL) protein receptors.

Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.

RiceMetaSys: Drought-miR, a one-stop solution for drought responsive miRNAs-mRNA module in rice.

MicroRNAs are key players involved in stress responses in plants and reports are available on the role of miRNAs in drought stress response in rice. This work reports the development of a database, RiceMetaSys: Drought-miR, based on the meta-analysis of publicly available sRNA datasets. From 28 drought stress-specific sRNA datasets, we identified 216 drought-responsive miRNAs (DRMs). The major features of the database include genotype-, tissue- and miRNA ID-specific search options and comparison of genotypes to identify common miRNAs. Co-localization of the DRMs with the known quantitative trait loci (QTLs), i.e., meta-QTL regions governing drought tolerance in rice pertaining to different drought adaptive traits, narrowed down this to 37 promising DRMs. To identify the high confidence target genes of DRMs under drought stress, degradome datasets and web resource on drought-responsive genes (RiceMetaSys: DRG) were used. Out of the 216 unique DRMs, only 193 had targets with high stringent parameters. Out of the 1081 target genes identified by Degradome datasets, 730 showed differential expression under drought stress in at least one accession. To retrieve complete information on the target genes, the database has been linked with RiceMetaSys: DRG. Further, we updated the RiceMetaSys: DRGv1 developed earlier with the addition of DRGs identified from RNA-seq datasets from five rice genotypes. We also identified 759 putative novel miRNAs and their target genes employing stringent criteria. Novel miRNA search has all the search options of known miRNAs and additionally, it gives information on their in silico validation features. Simple sequence repeat markers for both the miRNAs and their target genes have also been designed and made available in the database. Network analysis of the target genes identified 60 hub genes which primarily act through abscisic acid pathway and jasmonic acid pathway. Co-localization of the hub genes with the meta-QTL regions governing drought tolerance narrowed down this to 16 most promising DRGs. Database URL: http://14.139.229.201/RiceMetaSys_miRNA Updated database of RiceMetaSys URL: http://14.139.229.201/RiceMetaSysA/Drought/.

Genetic predisposition and high exposure to colistin in the early treatment period as independent risk factors for colistin-induced nephrotoxicity.

Colistin is known to cause nephrotoxicity due to its extensive reabsorption and accumulation in renal tubules. In vitro studies have identified the functional role of colistin transporters such as OCTN2, PEPT2, megalin, and P-glycoprotein. However, the role of these transporter gene variants in colistin-induced nephrotoxicity has not been studied. Utilizing targeted next-generation sequencing, we screened for genetic polymorphisms covering the colistin transporters (SLC15A1, SLC15A2, SLC22A5, LRP2, and ABCB1) in 42 critically ill patients who received colistimethate sodium. The genetic variants rs2257212 ((NM_021082.4):c.1048C>G) and rs13397109 ((NM_004525.3):C.7626C > T) were identified as being associated with an increased incidence of acute kidney injury (AKI) on Day 7. Colistin area under the curve (AUC) was predicted using a previously published pharmacokinetic model of colistin. Using logistic regression analysis, the predicted 24-h AUC of colistin was identified as an important contributor for increased odds of AKI on Day 7. Among 42 patients, 4 (9.5%) were identified as having high predisposition to colistin-induced AKI based on the presence of predisposing genetic variants. Determination of the presence of the abovementioned genetic variants and early therapeutic drug monitoring may reduce or prevent colistin-induced nephrotoxicity and facilitate dose optimization of colistimethate sodium.

Identification of DPYD variants and estimation of uracil and dihydrouracil in a healthy Indian population.

Aim: This study aimed to identify DPYD variants and the related but previously unexplored phenotype (plasma uracil, dihydrouracil [DHU], and the DHU-to-uracil ratio) in a healthy adult Indian population. Methods: Healthy adult volunteers (n = 100) had their uracil and DHU levels measured and were genotyped for selected variants. Results: Among the nine variants studied, c.1906-14763G>A and c.85T>C were the most prevalent. Participants with any of the variants except for c.85T>C and c.1627A>G had a significantly lower DHU-to-uracil ratio and those with c.1905+1G>A variant had significantly increased uracil concentration compared with wild-type. Conclusion: Participants with five variants were identified as having altered phenotypic measures, and 40% of the intermediate metabolizers had their phenotype in the terminal population percentiles.

Background: 5-fluorouracil (5-FU) is a medicine used in cancer treatment. It is eliminated from body by the enzyme DPD. Identifying deficiency in DPD before initiating 5-FU can save patients from oral, intestinal, and bone marrow toxic effects. Methods: The uracil and dihydrouracil (DHU, produced by DPD enzyme action) levels were measured and DPD gene (for identifying defects) was sequenced in 100 healthy adults. Results: Participants with DPD gene sequence that is known to be defective had higher plasma uracil levels and a low DHU-to-uracil ratio compared with those who did not have a defective gene. Conclusion: Measuring plasma uracil and DHU-to-uracil ratio can help identify people with defective DPD genes.

Model-Informed Rationale for Early Therapeutic Drug Monitoring of Colistin in Critically Ill Patients.

Adequate colistin exposure is important for microbiological clearance. This study was performed in critically ill patients >18 years old to develop a simplified nonparametric pharmacokinetic (PK) model of colistin for routine clinical use and to determine the role of dose optimization. The Non-Parametric Adaptive Grid algorithm within the Pmetrics software package for R was used to develop a PK model from 47 patients, and external validation of the final model was performed in 13 patients. A 1-compartment multiplicative gamma error model with 0-order input and first-order elimination of colistin was developed with creatinine clearance and serum albumin as covariates on elimination rate constant. An R2 for observed vs individual predicted colistin concentrations of 0.92 was obtained in the validation cohort. High interindividual variability in colistin steady-state area under the plasma concentration-time curve (AUC) from from 120 hours to 144 hours (coefficient of variation = 80.1%) and a high interoccasion variability (median coefficient of variation of AUC from time 0 to hours predicted every 8 hours for initial 96 hours after starting colistin = 23.8) was predicted in patients who received this antibiotic for a period of over 152 hours (n = 22). With the model-suggested dose regimen, only 20% of simulated profiles achieved AUC from time 0 to 24 hours in the range of 50 to 60 mg â€¢ h/L due to high variability in population PK. In this group of patients, steady-state colistin concentrations were predicted to be achieved >96 hours after initiation of colistimethate sodium. This study advocates the need for early and repeated therapeutic drug monitoring and dose optimization in critically ill patients to achieve adequate therapeutic concentration of colistin.

Metabolomics of early blight (Alternaria solani) susceptible tomato (Solanum lycopersicum) unfolds key biomarker metabolites and involved metabolic pathways.

Tomato (Solanum lycopersicum) is among the most important commercial horticultural crops worldwide. The crop quality and production is largely hampered due to the fungal pathogen Alternaria solani causing necrotrophic foliage early blight disease. Crop plants usually respond to the biotic challenges with altered metabolic composition and physiological perturbations. We have deciphered altered metabolite composition, modulated metabolic pathways and identified metabolite biomarkers in A. solani-challenged susceptible tomato variety Kashi Aman using Liquid Chromatography-Mass Spectrometry (LC-MS) based metabolomics. Alteration in the metabolite feature composition of pathogen-challenged (m/z 9405) and non-challenged (m/z 9667) plant leaves including 8487 infection-exclusive and 8742 non-infection exclusive features was observed. Functional annotation revealed putatively annotated metabolites and pathway mapping indicated their enrichment in metabolic pathways, biosynthesis of secondary metabolites, ubiquinone and terpenoid-quinones, brassinosteroids, steroids, terpenoids, phenylpropanoids, carotenoids, oxy/sphingolipids and metabolism of biotin and porphyrin. PCA, multivariate PLS-DA and OPLS-DA analysis showed sample discrimination. Significantly up regulated 481 and down regulated 548 metabolite features were identified based on the fold change (threshold ≥ 2.0). OPLS-DA model based on variable importance in projection (VIP scores) and FC threshold (> 2.0) revealed 41 up regulated discriminant metabolite features annotated as sphingosine, fecosterol, melatonin, serotonin, glucose 6-phosphate, zeatin, dihydrozeatin and zeatin-ß-D-glucoside. Similarly, 23 down regulated discriminant metabolites included histidinol, 4-aminobutyraldehyde, propanoate, tyramine and linalool. Melatonin and serotonin in the leaves were the two indoleamines being reported for the first time in tomato in response to the early blight pathogen. Receiver operating characteristic (ROC)-based biomarker analysis identified apigenin-7-glucoside, uridine, adenosyl-homocysteine, cGMP, tyrosine, pantothenic acid, riboflavin (as up regulated) and adenosine, homocyctine and azmaline (as down regulated) biomarkers. These results could aid in the development of metabolite-quantitative trait loci (mQTL). Furthermore, stress-induced biosynthetic pathways may be the potential targets for modifications through breeding programs or genetic engineering for improving crop performance in the fields.

Untargeted Metabolomics of Alternaria solani-Challenged Wild Tomato Species Solanum cheesmaniae Revealed Key Metabolite Biomarkers and Insight into Altered Metabolic Pathways.

Untargeted metabolomics of moderately resistant wild tomato species Solanum cheesmaniae revealed an altered metabolite profile in plant leaves in response to Alternaria solani pathogen. Leaf metabolites were significantly differentiated in non-stressed versus stressed plants. The samples were discriminated not only by the presence/absence of specific metabolites as distinguished markers of infection, but also on the basis of their relative abundance as important concluding factors. Annotation of metabolite features using the Arabidopsis thaliana (KEGG) database revealed 3371 compounds with KEGG identifiers belonging to biosynthetic pathways including secondary metabolites, cofactors, steroids, brassinosteroids, terpernoids, and fatty acids. Annotation using the Solanum lycopersicum database in PLANTCYC PMN revealed significantly upregulated (541) and downregulated (485) features distributed in metabolite classes that appeared to play a crucial role in defense, infection prevention, signaling, plant growth, and plant homeostasis to survive under stress conditions. The orthogonal partial least squares discriminant analysis (OPLS-DA), comprising a significant fold change (≥2.0) with VIP score (≥1.0), showed 34 upregulated biomarker metabolites including 5-phosphoribosylamine, kaur-16-en-18-oic acid, pantothenate, and O-acetyl-L-homoserine, along with 41 downregulated biomarkers. Downregulated metabolite biomarkers were mapped with pathways specifically known for plant defense, suggesting their prominent role in pathogen resistance. These results hold promise for identifying key biomarker metabolites that contribute to disease resistive metabolic traits/biosynthetic routes. This approach can assist in mQTL development for the stress breeding program in tomato against pathogen interactions.

Potential Use of Microbial Community Genomes in Various Dimensions of Agriculture Productivity and Its Management: A Review.

Agricultural productivity is highly influenced by its associated microbial community. With advancements in omics technology, metagenomics is known to play a vital role in microbial world studies by unlocking the uncultured microbial populations present in the environment. Metagenomics is a diagnostic tool to target unique signature loci of plant and animal pathogens as well as beneficial microorganisms from samples. Here, we reviewed various aspects of metagenomics from experimental methods to techniques used for sequencing, as well as diversified computational resources, including databases and software tools. Exhaustive focus and study are conducted on the application of metagenomics in agriculture, deciphering various areas, including pathogen and plant disease identification, disease resistance breeding, plant pest control, weed management, abiotic stress management, post-harvest management, discoveries in agriculture, source of novel molecules/compounds, biosurfactants and natural product, identification of biosynthetic molecules, use in genetically modified crops, and antibiotic-resistant genes. Metagenomics-wide association studies study in agriculture on crop productivity rates, intercropping analysis, and agronomic field is analyzed. This article is the first of its comprehensive study and prospects from an agriculture perspective, focusing on a wider range of applications of metagenomics and its association studies.

Analytical and clinical validation of dried blood spot and volumetric absorptive microsampling for measurement of tacrolimus and creatinine after renal transplantation.

BACKGROUND: Serial monitoring of tacrolimus and serum creatinine after renal transplantation is of vital importance. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the estimation of tacrolimus and creatinine, obtained from dried blood spots (DBS) or by volumetric absorptive microsampling (VAMS) was validated and the two sampling strategies were compared with traditional venous sampling. METHODS: The LC-MS/MS assay was validated using a shared extract for the estimation of tacrolimus and creatinine from DBS and VAMS independently. The relationship between the concentrations in DBS/VAMS specimens and in venous samples was assessed using Passing-Bablok (PB) analysis and the bias between the two methods was determined by the Bland Altman (BA) analysis. RESULTS: The imprecision and bias of tacrolimus and creatinine estimated from DBS and VAMS samples was <12% and was independent of the hematocrit (Hct). Samples were stable for five days at ambient temperature. From the PB regression analysis, correction equations were generated for the prediction of tacrolimus and creatinine values from DBS and VAMS samples. In a separate cohort of patients for validation, the corrected DBS and VAMS concentrations had a mean (95% CI) bias for tacrolimus of -0.64 (-2.98 to 1.70)% and -0.92 (-3.69 to 1.85)% respectively and for creatinine of 1.00 (-2.73 to 4.72)% and -0.71 (-3.74 to 2.32)% respectively. Using DBS and VAMS respectively, for tacrolimus, 91.8 and 89.8% of patient values and for creatinine, 69.4 and 81.6% of patient values were within the limits of clinical acceptance (within 15% agreement against the venous samples). CONCLUSION: We conclude that VAMS is the preferred single sampling option for estimating tacrolimus and creatinine in renal transplant patients.

Cyanobacteria-mediated phenylpropanoids and phytohormones in rice (Oryza sativa) enhance plant growth and stress tolerance.

Phenylpropanoids, flavonoids and plant growth regulators in rice (Oryza sativa) variety (UPR 1823) inoculated with different cyanobacterial strains namely Anabaena oryzae, Anabaena doliolum, Phormidium fragile, Calothrix geitonos, Hapalosiphon intricatus, Aulosira fertilissima, Tolypothrix tenuis, Oscillatoria acuta and Plectonema boryanum were quantified using HPLC in pot conditions after 15 and 30 days. Qualitative analysis of the induced compounds using reverse phase HPLC and further confirmation with LC-MS/MS showed consistent accumulation of phenolic acids (gallic, gentisic, caffeic, chlorogenic and ferulic acids), flavonoids (rutin and quercetin) and phytohormones (indole acetic acid and indole butyric acid) in rice leaves. Plant growth promotion (shoot, root length and biomass) was positively correlated with total protein and chlorophyll content of leaves. Enzyme activity of peroxidase and phenylalanine ammonia lyase and total phenolic content was fairly high in rice leaves inoculated with O. acuta and P. boryanum after 30 days. Differential systemic accumulation of phenylpropanoids in plant leaves led us to conclude that cyanobacterial inoculation correlates positively with plant growth promotion and stress tolerance in rice. Furthermore, the study helped in deciphering possible mechanisms underlying plant growth promotion and stress tolerance in rice following cyanobacterial inoculation and indicated the less explored avenue of cyanobacterial colonization in stress tolerance against abiotic stress.