Serine-rich domain of RNPS1 functions in activation of alternative splicing.

RNA-binding protein with serine-rich domain 1 (RNPS1) gets deposited on the mRNA during the process of splicing and concomitantly associates with the exon junction complex (EJC). RNPS1 participates in post-transcriptional gene regulation, including constitutive and alternative splicing, transcriptional regulation and nonsense-mediated mRNA decay. In this study, we found that the tethering of RNPS1 or its isolated serine-rich domain (S domain) causes exon inclusion of an HIV-1 splicing substrate. In contrast, overexpressing the RRM domain of RNPS1 acts in a dominant negative manner and leads to the exon skipping of endogenous apoptotic pre-mRNAs (Bcl-X and MCL-1). Further, tethering of core EJC proteins, eIF4A3, MAGOH, or Y14, does not lead to exon inclusion of an HIV substrate. Together, our results demonstrate how RNPS1 and its domains are differentially involved in alternative splicing activity.

Prevalence of self-medication practices among pregnant women in India: A systematic review and meta-analysis.

BACKGROUND: Self-medication practice among pregnant women is a global concern. However, its understanding in the Indian context is limited due to a lack of comprehensive studies. PURPOSE: This study aimed to comprehensively assess the prevalence of self-medication, the medications used for self-medication, diseases/conditions associated with self-medication, and the reasons for self-medication among Indian pregnant women. METHODS: This study was carried out following the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). A thorough search was done in PubMed, Embase, and Google Scholar to find articles that were published up until May 2023. Inclusion criteria comprised observational studies reporting self-medication prevalence among pregnant women in India. Data were extracted using a standardized sheet, and a random-effects model was applied to determine the overall prevalence of self-medication using R software. The I2 statistic was employed to assess the heterogeneity among the studies. RESULTS: This study analyzed eight studies with a collective sample size of 2208 pregnant women. The pooled prevalence of self-medication among pregnant Indian women was 19.3% (95% CI: 7.5%-41.3%; I2 = 99%; p < 0.01). Common self-treated conditions were cold, cough, fever, headache, and stomach disorders. Antipyretics, analgesics, antihistamines, and antacids were frequently used for self-medication. The perception of mild ailment, immediate alleviation, convenience, time savings, and advice from family, friends, or the media were all reasons for self-medication. Local pharmacies were the most usual source for obtaining drugs, and pharmacists, family, friends, and past prescriptions were common sources of medicine information. CONCLUSIONS: A low yet substantial number of pregnant women in India are engaged in self-medication practices. Appropriate strategies need to be planned to reduce self-medication practices to attain sustainable developmental goals for maternal health in India.

Delayed sowing and its ramifications: biophysical, yield and quality analysis of wheat cultivars in the northwest Indo-Gangetic plains.

BACKGROUND: The continuous cultivation of rice-wheat in the same field is a key element of double-cropping systems in the Indo-Gangetic plains. Yields of such cropping systems are increasingly challenged as climate change drives increases in temperature, terminal stress and uneven rainfall, delaying rice harvesting and subsequently delaying sowing of wheat. In this paper, we evaluate the optimum sowing dates to achieve high grain yield and quality of wheat cultivars in northwest India. Three cultivars of wheat, HD-2967, HD-3086 and PBW-723, were sown on three different dates at the research farm of ICAR-IARI, New Delhi, to generate different weather conditions at different phenological stages. Different biophysical attributes, photosynthetic rate, stomatal conductance and transpiration rate, were measured at different phenological stages. Yield and grain quality parameters such as protein, starch, amylopectin, amylose and gluten were measured in different cultivars sown on different dates. RESULTS: Biophysical parameters were found to be higher in timely sown crops followed by late-sown and very late-sown crops. Further, the different sowing dates had a significant (P < 0.05) impact on the grain quality parameters such as protein, starch, amylopectin, amylose and gluten content. Percentage increases in the value of starch and amylose content under timely sown were ~7% and 11.6%, ~5% and 8.4%, compared to the very late-sown treatment. In contrast, protein and amylopectin contents were found to increase by ~9.7% and 7.5%, ~13.8% and 16.6% under very late-sown treatment. CONCLUSION: High-temperature stress during the grain-filling periods significantly decreased the grain yield. Reduction in the grain yield was associated with a reduction in starch and amylose content in the grains. The protein content in the grains is less affected by terminal heat stress. Cultivar HD-3086 had higher growth, yield as well as quality parameters, compared to HD-2967 and PBW-723 in all treatments, hence could be adopted by farmers in northwest India. © 2024 Society of Chemical Industry.

Allergic contact dermatitis to lip care cosmetic products - a systematic review.

Aim: Lip care cosmetics products are any external preparation used by people to prevent drying, chapping, dullness, and beautification of lips. This study aimed to review the literature on allergic reactions induced by different types of lip care cosmetic products. Methods: A literature search was performed in PubMed from inception to June 2022. The study included articles published in English and available in full text. References of illegible articles were searched. Studies describing any patient who developed allergic contact dermatitis after the application of lip care cosmetic products were included. Results: A total of 47 reports consisting of 58 individuals experienced allergic reactions to lip care products. Several lip care cosmetics products, such as lipsticks, lip balms, lip salve, lip gloss, lip liner, and lip plumper, were found to be associated with allergic reactions. The most common ingredients that caused the allergic contact dermatitis were castor oil, benzophenone-3, gallate, wax, and colophony. Conclusions: Lip care cosmetics products contain several components that have been associated with allergic reactions. Awareness needs to be created among the general public and dermatologists regarding the presence of possible allergens in lip care cosmetic products.

A Study on Drug Prescribing Patterns in Vitiligo Patients of Andhra Pradesh in a Tertiary Care Teaching Hospital.

BACKGROUND: Vitiligo is a dermatological condition characterized by the appearance of white spots or patches on the skin due to the loss of skin pigment called melanocytes. The estimated prevalence of vitiligo is about 0.5-2% of the world population, but in India, the prevalence rate varies from 2 to 8%, depending on the region. This study aimed to assess drug prescribing patterns in vitiligo patients. MATERIALS AND METHODS: A prospective cross-sectional study was carried out in the Dermatology Department of Government General Hospital, Andhra Pradesh, India, from December 2019 to 2020. Patients aged ≥18 years, both genders, and diagnosed and receiving treatment for vitiligo were included in the study. All medicines prescribed to the patients were collected on the predesigned case report form. Ethical approval for this study was taken from the Institutional Ethics Committee of Rajiv Gandhi Institute of Medical Science (RIMS). The collected data were analyzed by using SPSS version 18. RESULTS: The most commonly prescribed class of drugs was corticosteroids (42.9%), followed by calcineurin inhibitors (13.4%), vitamins (14.6%), basic fibroblast growth factor (BFGF) (9.5%), moisturizers (6.9%), antihistamines (6.5%), and minerals (6.2%). Among corticosteroids, betamethasone was the most commonly prescribed drug, followed by clobetasol propionate. Topical drugs were prescribed more often than orally. CONCLUSION: The prescription pattern in vitiligo patients is as per the guidelines and recommendations. However, further studies using multiple centers are recommended to verify our findings.

Rice WRKY13 TF protein binds to motifs in the promoter region to regulate downstream disease resistance-related genes.

In plants, pathogen resistance is brought about by the binding of certain transcription factor (TF) proteins to the cis-elements of certain target genes. These cis-elements are present upstream in the motif of the promoters of each gene. This ensures the binding of a specific TF to a specific promoter, therefore regulating the expression of that gene. Therefore, the study of each promoter sequence of all the rice genes would help identify the target genes of a specific TF. Rice 1 kb upstream promoter sequences of 55,986 annotated genes were analyzed using the Perl program algorithm to detect WRKY13 binding motifs (bm). The resulting genes were grouped using Gene Ontology and gene set enrichment analysis. A gene with more than 4 TF bm in their promoter was selected. Ten genes reported to have a role in rice disease resistance were selected for further analysis. Cis-acting regulatory element analysis was carried out to find the cis-elements and confirm the presence of the corresponding motifs in the promoter sequences of these genes. The 3D structure of WRKY13 TF and the corresponding ten genes were built, and the interacting residues were determined. The binding capacity of WRKY13 to the promoter of these selected genes was analyzed using docking studies. WRKY13 was considered for docking analysis based on the prior reports of autoregulation. Molecular dynamic simulations provided more details regarding the interactions. Expression data revealed the expression of the genes that helped provide the mechanism of interaction. Further co-expression network helped to characterize the interaction of these selected disease resistance-related genes with the WRKY13 TF protein. This study suggests downstream target genes that are regulated by the WRKY13 TF. The molecular mechanism involving the gene network regulated by WRKY13 TF in disease resistance against rice fungal pathogens is explored.

RNPS1 functions as an oncogenic splicing factor in cervical cancer cells.

Numerous recent studies suggest that cancer-specific splicing alteration is a critical contributor to the pathogenesis of cancer. RNA-binding protein with serine-rich domain 1, RNPS1, is an essential regulator of the splicing process. However, the defined role of RNPS1 in tumorigenesis still remains elusive. We report here that the expression of RNPS1 is higher in cervical carcinoma samples from The Cancer Genome Atlas (TCGA-cervical squamous cell carcinoma and endocervical adenocarcinoma) compared to the normal tissues. Consistently, the expression of RNPS1 was high in cervical cancer cells compared to a normal cell line. This study shows for the first time that RNPS1 promotes cell proliferation and colony-forming ability of cervical cancer cells. Importantly, RNPS1 positively regulates migration-invasion of cervical cancer cells. Intriguingly, depletion of RNPS1 increases the chemosensitivity against the chemotherapeutic drug doxorubicin in cervical cancer cells. Further, we characterized the genome-wide isoform switching stimulated by RNPS1 in cervical cancer cell. Mechanistically, RNA-sequencing analysis showed that RNPS1 regulates the generation of tumor-associated isoforms of key genes, particularly Rac1b, RhoA, MDM4, and WDR1, through alternative splicing. RNPS1 regulates the splicing of Rac1 pre-mRNA via a specific alternative splicing switch and promotes the formation of its tumorigenic splice variant, Rac1b. While the transcriptional regulation of RhoA has been well studied, the role of alternative splicing in RhoA upregulation in cancer cells is largely unknown. Here, we have shown that the knockdown of RNPS1 in cervical cancer cells leads to the skipping of exons encoding the RAS domain of RhoA, consequently causing decreased expression of RhoA. Collectively, we conclude that the gain of RNPS1 expression may be associated with tumor progression in cervical carcinoma. RNPS1-mediated alternative splicing favors an active Rac1b/RhoA signaling axis that could contribute to cervical cancer cell invasion and metastasis. Thus, our work unveils a novel role of RNPS1 in the development of cervical cancer.

Current trends in the development of soy-based foods containing probiotics and paving the path for soy-synbiotics.

In the world of highly processed foods, special attention is drawn to the nutrient composition and safety of consumed food products. Foods fortified with probiotic bacteria confer beneficial effects on human health and are categorized as functional foods. The salubrious activities of probiotics include the synthesis of vital bioactives, prevention of inflammatory diseases, anticancerous, hypocholesterolemic, and antidiarrheal effects. Soy foods are exemplary delivery vehicles for probiotics and prebiotics and there are diverse strategies to enhance their functionality like employing mixed culture fermentation, engineering probiotics, and incorporating prebiotics in fermented soy foods. High potential is ascribed to the concurrent use of probiotics and prebiotics in one product, termed as "synbiotics," which implicates synergy, in which a prebiotic ingredient particularly favors the growth and activity of a probiotic micro-organism. The insights on emended bioactive profile, metabolic role, and potential health benefits of advanced soy-based probiotic and synbiotic hold a promise which can be profitably implemented to meet consumer needs. This article reviews the available knowledge about strategies to enhance the nutraceutical potential, mechanisms, and health-promoting effects of advanced soy-based probiotics. Traditional fermentation merged with diverse strategies to improve the efficiency and health benefits of probiotics considered vital, are also discussed.

Adverse events of clenbuterol among athletes: a systematic review of case reports and case series.

Clenbuterol is a potent beta-2 agonist widely misused by professional athletes and bodybuilders. Information on clenbuterol associated adverse events is present in case reports and case series, though it may not be readily available. This systematic review aimed to critically evaluate the evidence of adverse events associated with clenbuterol among athletes. The search strategy was in accordance with PRISMA guidelines. Databases such as PubMed, Science Direct, Scopus, and Google Scholar were searched from 1990 to October 2021 to find out the relevant case reports and case series. There were 23 included studies. Using a suitable scale, the included studies' methodological quality analysis was evaluated. In total, 24 athletes experienced adverse events. Oral ingestion of clenbuterol was the most preferred route among them. The daily administered dose of clenbuterol was ranging from 20 µg to 30 mg. Major adverse events experienced by athletes were supraventricular tachycardia, atrial fibrillation, hypotension, chest pain, myocardial injury, myocarditis, myocardial ischemia, myocardial infarction, cardiomyopathy, hepatomegaly, hyperglycemia, and death. The cardiac-related complications were the most commonly occurring adverse events. Clenbuterol is notorious to produce life-threatening adverse events including death. Lack of evidence regarding the performance-enhancing effects of clenbuterol combined with its serious toxicities questions the usefulness of this drug in athletes.

Tissue heterogeneity of mitochondrial activity, biogenesis and mitochondrial protein gene expression in buffalo.

BACKGROUND: Cellular metabolism is most invariant process, occurring in all living organisms, which involves mitochondrial proteins from both nuclear and mitochondrial genomes. The mitochondrial DNA (mtDNA) copy number, protein-coding genes (mtPCGs) expression, and activity vary between various tissues to fulfill specific energy demands across the tissues. METHODS AND RESULTS: In present study, we investigated the OXPHOS complexes and citrate synthase activity in isolated mitochondria from various tissues of freshly slaughtered buffaloes (n = 3). Further, the evaluation of tissue-specific diversity based on the quantification of mtDNA copy numbers was performed and also comprised an expression study of 13 mtPCGs. We found that the functional activity of individual OXPHOS complex I was significantly higher in the liver compared to muscle and brain. Additionally, OXPHOS complex III and V activities was observed significantly higher levels in liver compared to heart, ovary, and brain. Similarly, CS-specific activity differs between tissues, with the ovary, kidney, and liver having significantly greater. Furthermore, we revealed the mtDNA copy number was strictly tissue-specific, with muscle and brain tissues exhibiting the highest levels. Among 13 PCGs expression analyses, mRNA abundances in all genes were differentially expressed among the different tissue. CONCLUSIONS: Overall, our results indicate the existence of a tissue-specific variation in mitochondrial activity, bioenergetics, and mtPCGs expression among various types of buffalo tissues. This study serves as a critical first stage in gathering vital comparable data about the physiological function of mitochondria in energy metabolism in distinct tissues, laying the groundwork for future mitochondrial based diagnosis and research.

Functional role of SAP18 protein: From transcriptional repression to splicing regulation.

Sin3 associated protein 18 (SAP18) is an evolutionary conserved protein, originally discovered in a complex with the transcriptional regulatory protein, Sin3. Subsequent investigations revealed SAP18 as an integral splicing component of the exon junction complex (EJC)-associated apoptosis-and splicing-associated protein (ASAP)/PNN-RNPS1-SAP18 (PSAP) complex. In association with Sin3, SAP18 contributes toward transcriptional repression of genes implicated in embryonic development, stress response, human immunodeficiency virus type 1 replication, and tumorigenesis. As a part of EJC, SAP18 mediates alternative splicing events and suppresses the cryptic splice sites present within flanking regions of exon-exon junctions. In this review, we provide a thorough discussion on SAP18, focussing on its conserved dual role in transcriptional regulation and messenger RNA splicing. Recent research on the involvement of SAP18 in the emergence of cancer and human disorders has also been highlighted. The potential of SAP18 as a therapeutic target is also discussed in these recent studies, particularly related to malignancies of the myeloid lineage.

Isoflavones Play a Potential Role in Off-Flavour Scavenging, with a Key Role of IFS2 in Isoflavone Accumulation in Soybean Seeds.

Research background: Soybean (Glycine max (L.) Merr) is a nutrient-rich crop with a high protein content and various bioactive compounds with health-promoting properties. Nevertheless, it is poorly accepted as a food by consumers due to its off-flavour. Due to the ubiquitous presence of isoflavones in soybeans, their inherent antioxidant potential and inhibitory effect on lipoxygenase activity, their sensory properties are currently being considered to mitigate the off-flavour. Experimental approach: In the present study, the content and composition of isoflavones in 17 soybean cultivars are determined. The correlation between the isoflavone mass fraction and lipid peroxidation was also established, using thiobarbituric acid (TBA) value and carbonyl compound concentration as indices for the development of off-flavour. Cloning, gene expression analysis and in silico analysis of isoflavone synthase isoforms (IFS1 and IFS2) were also performed. Results and conclusions: The total isoflavone mass fraction in soybean genotypes ranged from (153.5±7.2) µg/g for PUSA 40 to (1146±43) µg/g for Bragg. There was a moderately negative correlation between the indices of off-flavour formation and the genistein/daidzein ratio (p<0.1). However, the correlation with total isoflavone mass fraction was found to be insignificant, indicating complex interactions. Higher protein-protein interactions for the predicted structure of IFS2 with other biosynthesis enzymes and its comparatively higher expression in the Bragg than that of IFS1 indicated its more important role in isoflavone synthesis. Novelty and scientific contribution: The genistein/daidzein mass ratio was found to be an important factor in controlling off-flavour. IFS2 was identified as key to produce soybeans with high isoflavone mass fraction and potentially lower off-flavour formation.

Prediction of glaciated area fraction over the Sikkim Himalayan Region, India: a comparative study using response surface method, random forest, and artificial neural network.

Glacier area fraction at high altitude mountains is a serious worry in today's time triggered by climate change. The current information on this natural resource is very important for the survival of humanity as it affects the water, food, and energy security of people dependent on it. Due to its problematic accessibility and tough environmental condition, ground monitoring is quite challenging. This study investigates the impact of environmental parameters and pollutants on glacier area fraction over the Eastern Himalaya region and its prediction through random forest (RF), multilayer perceptron (MLP), radial basis function analysis (RBFN), and response surface methodology (RSM) models. The data are obtained from the Goddard Earth Sciences Data and Information Services Center (GES DISC), NASA's data archive portal ( https://giovanni.gsfc.nasa.gov ). The collinearity of independent variables reveals that all selected input parameters are highly correlated with R2 value > 0.9. The RSM and RF model provided valuable insight of the predictor's significance in addition to their capability to predict the response. The model performance was evaluated in terms of R2 value and the error matrices. The model's R2 value was found to be 0.843, 0.839, 0.838, and 0.743 for MLP, RBFN, RF, and RSM respectively. Although, the neural network model R2 values are the highest, but the most reliable and suitable model is RF as the error matrices for this model are much lower than others. This study encourages the investigation of the hybridization of these models for more accurate prediction.

Barriers to the effective management and prevention of post kala-azar dermal leishmaniasis (PKDL) in the Indian subcontinent.

Post kala-azar dermal leishmaniasis (PKDL) is a skin disease that usually occurs among individuals with a past history of visceral leishmaniasis (VL). PKDL cases act as a reservoir of parasites and may play a significant role in disease transmission. Hence, prompt detection and complete treatment of PKDL cases are crucial for the control and elimination of VL. The purpose of this review was to highlight the barriers to effective control and prevention of VL/PKDL as well as potential solutions in India. Main obstacles are lack of knowledge about the disease and its vector, poor treatment-seeking behaviours, ineffective vector control measures, lack of confirmatory diagnostics in endemic areas, limited drug choices, treatment noncompliance among patients, drug resistance, and a lack of an adequate number of trained personnel in the health system. Therefore, in order to control and successfully eliminate VL in the Indian subcontinent, early detection of PKDL cases, improved diagnosis and treatment, raising awareness, and effective vector control mechanisms are necessary.

Diabetes induces dysregulation of microRNAs associated with survival, proliferation and self-renewal in cardiac progenitor cells.

AIMS/HYPOTHESIS: Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. METHODS: CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). RESULTS: Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. CONCLUSIONS/INTERPRETATION: Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

Conserved miRNAs modulate the expression of potential transcription factors of isoflavonoid biosynthetic pathway in soybean seeds.

Despite the significant importance of soybean isoflavone, the regulatory mechanism of miRNAs during its biosynthesis is highly unexplored. In the present work, nine existing miRNAs along with their ten corresponding target genes were identified and validated in soybean for their possible role during isoflavonoid biosynthesis and accumulation. Temporal expression analysis at four key stages of seed development (35, 45, 55 and 65DAF) of all the miRNA-target pairs showed varying degree of differential accumulation in two soybean genotypes (NRC37: high isoflavone; and NRC7: low isoflavone). Differential expression of MYB65-Gma-miR159, MYB96-Gma-miRNA1534, MYB176-Gma-miRNA5030, SPL9-Gma-miRNA156, TCP3, TCP4-Gma-miRNA319, WD40-Gma-miRNA162, UDP-glucose: flavonoid 3-O-glucosyltransferase-Gma-miRNA396, and CHI3-Gma-miRNA5434 showed an important relationship with their targets in both the soybean genotypes across all the stages. Therefore, the finding of the present work would certainly increase our understanding of molecular regulation of isoflavone biosynthetic pathway mediated by the miRNA which would guide molecular breeder to develop isoflavone rich soybean cultivars.

Genomic organization, phylogenetic comparison, and expression profiles of the SPL family genes and their regulation in soybean.

SQUAMOSA Promoter-Binding Protein-Like (SPL) genes form a major family of plant-specific transcription factors and play an important role in plant growth and development. In this study, we report the identification of 41 SPL genes (GmSPLs) in the soybean genome. Phylogenetic analysis revealed that these genes were divided into five groups (groups 1-5). Further, exon/intron structure and motif composition revealed that the GmSPL genes are conserved within their same group. The N-terminal zinc finger 1 (Zn1) of the SBP domain was a CCCH (Cys3His1) and the C terminus zinc finger 2 (Zn2) was a CCHC (Cys2HisCys) type. The 41 GmSPL genes were distributed unevenly on 17 of the 20 chromosomes, with tandem and segmental duplication events. We found that segmental duplication has made an important contribution to soybean SPL gene family expansion. The Ka/Ks ratios revealed that the duplicated GmSPL genes evolved under the effect of purifying selection. In addition, 17 of the 41 GmSPLs were found as targets of miR156; these might be involved in their posttranscriptional regulation through miR156. Importantly, RLM-RACE analysis confirmed the GmmiR156-mediated cleavage of GmSPL2a transcript in 2-4 mm stage of soybean seed. Alternative splicing events in 9 GmSPLs were detected which produces transcripts and proteins of different lengths that may modulate protein signaling, binding, localization, stability, and other properties. Expression analysis of the soybean SPL genes in various tissues and different developmental stages of seed suggested distinct spatiotemporal patterns. Differences in the expression patterns of miR156-targeted and miR156-non-targeted soybean SPL genes suggest that miR156 plays key functions in soybean development. Our results provide an important foundation for further uncovering the crucial roles of GmSPLs in the development of soybean and other biological processes.

Molecular cloning, heterologous expression and functional characterization of gamma tocopherol methyl transferase (γ-TMT) from Glycine max.

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74-87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded ß sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni2+-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules.