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.

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.

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.

Fungal Genomic Resources for Strain Identification and Diversity Analysis of 1900 Fungal Species.

Identification and diversity analysis of fungi is greatly challenging. Though internal transcribed spacer (ITS), region-based DNA fingerprinting works as a "gold standard" for most of the fungal species group, it cannot differentiate between all the groups and cryptic species. Therefore, it is of paramount importance to find an alternative approach for strain differentiation. Availability of whole genome sequence data of nearly 2000 fungal species are a promising solution to such requirement. We present whole genome sequence-based world's largest microsatellite database, FungSatDB having >19M loci obtained from >1900 fungal species/strains using >4000 assemblies across globe. Genotyping efficacy of FungSatDB has been evaluated by both in-silico and in-vitro PCR. By in silico PCR, 66 strains of 8 countries representing four continents were successfully differentiated. Genotyping efficacy was also evaluated by in vitro PCR in four fungal species. This approach overcomes limitation of ITS in species, strain signature, and diversity analysis. It can accelerate fungal genomic research endeavors in agriculture, industrial, and environmental management.

Stage-dependent concomitant microbial fortification improves soil nutrient status, plant growth, antioxidative defense system and gene expression in rice.

Stage-dependent concomitant fortification of rice (Oryza sativa L.) varieties PB1612 and CO51 with microbial inoculants Trichoderma asperellum and Pseudomonas fluorescens as seed coating, seedling root inoculation and soil application enhanced growth, activated antioxidant enzymes and modulated defence-related genes in plants. Microbial inoculants improved shoot height, tiller numbers, fresh weight and dry biomass. Co-inoculation was more impactful in enhancing plant growth and development as compared to single inoculation. Single and co-inoculation improved organic carbon (OC) and N, P and K content in the soil substantially. Mean values between control and co-inoculation varied significantly for OC in PB1612 (p0.001) and CO51 (p0.019) and phosphorus content in PB1612 (p0.044) and CO51 (p0.021). Microbial inoculation enhanced soil nutrients and increased their bioavailability for the plants. Total polyphenolics, flavonoids and protein content increased in the leaves following microbial inoculation. Enhanced non-enzymatic antioxidant parameters (ABTS, DPPH, Fe-ion reducing power and Fe-ion chelation) was found in microbe inoculated rice reflecting high free radical scavenging activity in polyphenolics-rich leaf extracts. Increased enzyme activity of superoxide dismutase (SOD), glutathione reductase (GR), phenylalanine ammonia-lyase (PAL), peroxidase (PO), glutathione peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT) showed improved ROS scavenging in rice plants having co-inoculation. Over-expression of PAL, cCuZn-SOD and CAT genes in microbial inoculated rice plants was recorded. The study concludes that plant stage-wise concomitant fortification by microbial inoculants could play multi-pronged manifestations at physiological, biochemical and molecular level in rice to positively influence growth, development and defense attributes in plants.

Microbial inoculation in rice regulates antioxidative reactions and defense related genes to mitigate drought stress.

Microbial inoculation in drought challenged rice triggered multipronged steps at enzymatic, non-enzymatic and gene expression level. These multifarious modulations in plants were related to stress tolerance mechanisms. Drought suppressed growth of rice plants but inoculation with Trichoderma, Pseudomonas and their combination minimized the impact of watering regime. Induced PAL gene expression and enzyme activity due to microbial inoculation led to increased accumulation of polyphenolics in plants. Enhanced antioxidant concentration of polyphenolics from microbe inoculated and drought challenged plants showed substantially high values of DPPH, ABTS, Fe-ion reducing power and Fe-ion chelation activity, which established the role of polyphenolic extract as free radical scavengers. Activation of superoxide dismutase that catalyzes superoxide (O2-) and leads to the accumulation of H2O2 was linked with the hypersensitive cell death response in leaves. Microbial inoculation in plants enhanced activity of peroxidase, ascorbate peroxidase, glutathione peroxidase and glutathione reductase enzymes. This has further contributed in reducing ROS burden in plants. Genes of key metabolic pathways including phenylpropanoid (PAL), superoxide dismutation (SODs), H2O2 peroxidation (APX, PO) and oxidative defense response (CAT) were over-expressed due to microbial inoculation. Enhanced expression of OSPiP linked to less-water permeability, drought-adaptation gene DHN and dehydration related stress inducible DREB gene in rice inoculated with microbial inoculants after drought challenge was also reported. The impact of Pseudomonas on gene expression was consistently remained the most prominent. These findings suggested that microbial inoculation directly caused over-expression of genes linked with defense processes in plants challenged with drought stress. Enhanced enzymatic and non-enzymatic antioxidant reactions that helped in minimizing antioxidative load, were the repercussions of enhanced gene expression in microbe inoculated plants. These mechanisms contributed strongly towards stress mitigation. The study demonstrated that microbial inoculants were successful in improving intrinsic biochemical and molecular capabilities of rice plants under stress. Results encouraged us to advocate that the practice of growing plants with microbial inoculants may find strategic place in raising crops under abiotic stressed environments.