Roshan Kumar

Vascular plants rapidly coordinate root and shoot responses to water stress. Abscisic acid (ABA) mediates these adaptations; however, it remains unclear which cells produce ABA, whether ABA synthesis shifts during stress, and whether ABA movement is required for its adaptive functions. Here, we map ABA biosynthesis at cellular resolution in Arabidopsis and report that water-stress adaptive responses in roots and shoots require movement of ABA and its precursor AB-aldehyde from vascular tissues to target cells. We suggest that ABA accumulation arises from two parallel routes: (i) ABA synthesized in the vasculature via ABA2 and AAO3, then moving to guard cells, and (ii) phloem-derived AB-aldehyde being converted to ABA in the epidermis or bundle sheath by AAO1 and AAO2. Finally, we predict that tightly packed cells beneath leaf veins facilitate efficient ABA delivery to guard cells, an anatomical arrangement that has enabled angiosperms to evolve the use of ABA to rapidly close stomata.

Antibiotic self-medication (ASM) significantly contributes to antimicrobial resistance (AMR), a pressing global health concern. However, the impact of knowledge, health information access, and health-seeking behavior on ASM in Thailand is poorly understood. This study aimed to evaluate these influences using data from the 2021 health and welfare survey conducted by Thailand’s National Statistical Office. A generalized linear mixed model was applied to assess associations. Variables with a p-value <0.25 in bivariate analysis were further analyzed in a multivariable model, with adjusted odds ratios (AOR), 95% confidence intervals (CI), and p-values reported. Among 26,030 participants, 2.79% (95% CI: 2.59%-2.99%) engaged in ASM. Low knowledge of antibiotic use was strongly linked to ASM (AOR=4.30, 95% CI: 2.61-7.08), as was moderate knowledge (AOR=2.47, 95% CI: 1.49-4.09). Lack of antibiotic-related information significantly increased ASM (AOR=3.86, 95% CI: 3.03-4.92). Recent health-seeking behaviors, including over-the-counter medication (AOR=3.54, 95% CI: 2.02-6.21) and avoiding public and private hospitals, were also major contributors. Medication and transportation expenses were additional significant factors. Enhancing access to health information, knowledge, and healthcare services could reduce ASM in Thailand.

In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation of Leu flux upon removing the IPMS regulatory domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu–Val homeostasis in plants.

Diabetes mellitus (DM) and its associated complications present a major health problem for the Thai people, including Buddhist monks. This study aimed to identify the influence of behavioural and genetic variables on the prevalence of type 2 diabetes mellitus in middle-aged monks of Thailand. The study analyzed data from the health data centre of the Department of Public Health, Thailand, focusing on middle-aged monks in 76 provinces across 12 regions. The study recruited 6,408 Thai monks who met inclusion and exclusion criteria between October 2015-November 2019. Multivariable analysis was performed using a generalized linear mixed model, with adjusted odds ratios and 95% confidence intervals reported at a significance level of 0.05. Our study found that among the middle-aged monks of Thailand, 7.12% (95% CI: 6.49-7.78) had diabetes mellitus out of a total of 6,408. The results of our multivariable analysis showed that the following factors were associated with diabetes mellitus in monks of Thailand: respondents’ body mass index (BMI) ≥30 kg/m2 (AOR=4.26, 95% CI: 3.08-5.90, p-value <0.001), respondents with a family history of DM (AOR=3.37, 95% CI: 2.52-4.81, p-value<0.001), and respondent’s age ranges of 55-59 (AOR=2.87, 95% CI: 2.26-3.84, p-value<0.001), respectively. Diabetes millatus is significantly associated with the respondents who were obese, aged, and had a family history of DM. Therefore, a regular DM screening program as well as a timely health monitoring system for monks will enhance the diabetes control program in reducing the DM burden in Thailand.

In the leucine biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by binding of the end-product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In - vitro studies showed that removal of the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation upon removal of the IPMS C-terminal domain on the Leu flux have not been previously explored in plants. Here, we show that in the absence of its regulatory domain, an unexpected alternative regulatory loop acts to control plant IPMS catalysis. Removal of IPMS regulatory domain using CRISPR/Cas9 significantly reduced the formation of end-product Leu in - planta , but increased the levels of Leu pathway intermediates. Additionally, delayed growth was observed when IPMS devoid of regulatory domain was introduced into IPMS -null mutants of E. coli and Arabidopsis . Combining the metabolomic and biochemical analysis, we found that the Leu pathway intermediate, α-ketoisocaproate, was a competitive inhibitor of IPMS with a truncated regulatory domain. Thus, we demonstrate that the C-terminal regulatory domain of IPMS is biologically favored since it maintains Leu homeostasis while bypassing the possibility of competitive inhibition by a pathway intermediate. Significance: Till date it was known that the limited profile of essential amino acid-leucine in plants, is majorly due to the feedback inhibition of its pathway enzyme, IPMS, by the binding of accumulating leucine at its regulatory domain. So, can we increase the leucine pool in plants by removing the IPMS regulatory domain? Herein we show that, the targeted removal of this domain under native condition had led to low leucine pool and compromised growth phenotype but observed an accumulation in the leucine pathway intermediates. We uncover a hidden function of the IPMS regulatory domain to avoid an intermediate inhibition on IPMS activity, which could limit the end-product. The study brings an unknown regulatory checkpoint in maintaining leucine homeostasis in plants.

Summary: Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR‐edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR‐edited lines displayed a defence response at par or better than that of the wild‐type line. The GTR‐edited lines were equivalent to the wild‐type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low‐seed, high‐leaf glucosinolate lines with an uncompromised defence and yield.

This cross-sectional study identified the determinants and barriers of pre- hospital emergency medical services (EMS) utilization among general critical emergency patients in Thailand, using the data of general critical emergency patients (red zone) from the information technology for emergency medical system (ITEMS) of the National Emergency Medical Service Institute. The sample of 889 critical emergency patients was selected through multistage random sampling method. Data were analyzed by multiple logistic regression. The findings indicated that 41.51% of general critical emergency patients used EMS through the EMS rescue hotline system of the National Institute of Emergency Medicine in which most of them were diabetic patients (24.75%). The determinants relating to EMS utilization of general critical emergency patients consisted of level of knowledge on EMS system (AdjOR: 5.77; 95%CI: 2.8-11.87), confidence in the safety of service utilization (AdjOR: 5.04; 95%CI: 3.65-6.98), recognition in service and severity of illness (AdjOR: 3.22; 95%CI: 2.17-4.76), service adequacy (AdjOR: 1.92; 95%CI: 1.41-2.63), educational background (AdjOR:1.69; 95%CI: 1.13-2.53), and service satisfaction (AdjOR: 1.14; 95%CI: 1.07-1.21). Nearly half of the people were utilizing the EMS services in Thailand. However, required level of knowledge on EMS, confidence on safety on service utilization, service adequacy as well as patient’s educational background had tremendous role on the proper utilization of EMS.

Glucosinolate content in the Brassica oilseed crops has been reduced to the globally accepted Canola quality levels, making the seed meal useful as a protein-rich animal feed. However, the overall lower glucosinolate content in such plants renders them vulnerable to pests. We report CRISPR/Cas9-based editing of glucosinolate transporter ( GTR ) family genes to develop ideal oilseed mustard lines with the desired low levels of seed glucosinolates while maintaining high glucosinolate levels in leaves and pod walls, without affecting the seed yield and seed quality traits. The low-seed and high-leaf glucosinolate mustard lines with improved nutritional and defense traits will raise its international trade potential both as oilseed and feed crop.

Key message Two BjuGα proteins exhibit conserved GTP-binding and GTP-hydrolysis activities, and function in maintaining overall plant architecture and controlling multiple yield-related traits in the oilseed Brassica juncea. Abstract Heterotrimeric G-protein (Gα, Gβ and Gγ) are key signal transducers, well characterized in model plants Arabidopsis and rice. However, our knowledge about the roles played by G-proteins in regulating various growth and developmental traits in polyploid crops, having a complex G-protein signalling network, is quite sparse. In the present study, two Gα encoding genes (BjuA.Gα1 and BjuB.Gα1) were isolated from the allotetraploid Brassica juncea, a globally cultivated oilseed crop of the Brassicaceae family. BjuGα1 genes share a close evolutionary relationship, and the encoded proteins exhibit highly conserved G-protein activities while showing expression differentiation, wherein BjuA.Gα1 was the highly abundant transcript during plant growth and developmental stages. RNAi based suppression of BjuGα1 displayed compromised effects on most of the tested vegetative and reproductive parameters, particularly plant height (32–58%), flower and siliques dimensions, and seed weight (11–13%). Further, over-expression of a constitutively active Gα, lacking the GTPase activity, produced plants with increased height, organ size and seed weight (7–25%), without altering seed quality traits like fatty acid composition, glucosinolates, oil and protein contents. Our study demonstrates that BjuGα1 proteins control overall plant architecture and multiple yield-related traits in the oilseed B. juncea, suggesting that BjuGα1 could be a promising target for crop improvement.

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection.