MPK4-mediated phosphorylation of PHYTOCHROME INTERACTING FACTOR4 controls thermosensing by regulating histone variant H2A.Z deposition.

Plants can perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis is well established, although the complete regulatory pathway involved in thermosensing remains elusive. We establish that an increase in temperature from 22 to 28 °C induces upregulation and activation of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) in Arabidopsis (Arabidopsis thaliana), subsequently leading to the phosphorylation of PIF4. Phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6), which is required for mediating the deposition of histone variant H2A.Z at its target loci. Furthermore, we demonstrate that variations in ARP6 expression in PIF4 phosphor-null and phosphor-mimetic seedlings affect hypocotyl growth at 22 and 28 °C by modulating the regulation of ARP6-mediated H2A.Z deposition at the loci of genes involved in elongating hypocotyl cells. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in a temperature-dependent manner. Taken together, these findings highlight the regulatory mechanism of thermosensing by which MPK4-mediated phosphorylation of PIF4 affects ARP6-mediated H2A.Z deposition at the genes involved in hypocotyl cell elongation.

The small RNA biogenesis in rice is regulated by MAP kinase-mediated OsCDKD phosphorylation.

CDKs are the master regulator of cell division and their activity is controlled by the regulatory subunit cyclins and phosphorylation by the CAKs. However, the role of MAP kinases in regulating plant cell cycle or CDKs have not been explored. Here, we report that the MAP kinases OsMPK3, OsMPK4, and OsMPK6 physically interact and phosphorylate OsCDKD and its regulatory subunit OsCYCH in rice. MAP kinases phosphorylate CDKD at Ser-168 and Thr-235 residues in OsCDKD. The MAP kinase-mediated phosphorylation of OsCDKD is required for its activation to control the small RNA biogenesis. The phosphodead version of OsCDKD fails to activate the C-terminal domain of RNA Polymerase II, thereby negatively impacting small RNA transcription. Further, the overexpression lines of wild-type (WT) OsCDKD and phosphomimic OsCDKD show increased root growth, plant height, tiller number, panicle number, and seed number in comparison to WT, phosphodead OsCDKD-OE, and kinase-dead OsCDKD-OE plants. In a nutshell, our study establishes a novel regulation of OsCDKD by MAPK-mediated phosphorylation in rice. The phosphorylation of OsCDKD by MAPKs imparts a positive effect on rice growth and development by regulating miRNAs transcription.

Electro-organic synthesis of isatins and hydrazones through C-N cross-coupling and C(sp2)-H/C(sp3)-H functionalization.

An efficient and unique approach to synthesize isatin (indole-2,3-dione) from 2-aminoacetophenone under electrochemical conditions supported by I2-DMSO through C-N cross-coupling and C(sp2)-H/C(sp3)-H functionalization is presented. This synthetic method spans a wide range of substituted 2-aminoacetophenone substrates. The use of iodine as a promoter and shorter reaction times produced good to very good yields of isatin derivatives, which is a significant improvement over the reaction in a batch process. Further, hydrazones of isatin were synthesized by using hydrazine hydrate which produces electrochemically active molecules, namely isatin-hydrazones. The hydrazones of acetophenone were also obtained using the same reaction protocol. Additionally, the effect of increasing scan rate studied using cyclic voltammetry shows that the process followed a diffusion-controlled mechanism.

Regulation of tapetum-specific A9 promoter by transcription factors AtMYB80, AtMYB1 and AtMYB4 in Arabidopsis thaliana and Nicotiana tabacum.

Tapetum-specific promoters have been successfully used for developing transgenic-based pollination control systems. Although several tapetum-specific promoters have been identified, in-depth studies on regulation of such promoters are scarce. The present study analyzes the regulation of the A9 promoter, one of the first tapetum-specific promoter identified in Arabidopsis thaliana. Transcription factors (TFs) AtMYB80, AtMYB1 (positive regulators) identified by in silico analysis were found to upregulate A9 promoter activity following the over-expression of the TFs in transient and stable (transgenic) expression assays in both A. thaliana and tobacco. Furthermore, mutations of binding sites of these TFs in the A9 promoter led to loss of its activity. The role of a negative regulator AtMYB4 was also studied by analyzing the activity of A9 promoter following transient expression of RNAi against the TF and by mutating binding sites for AtMYB4 in the A9 promoter. While no changes were observed in case of A. thaliana, the A9 promoter was activated in the roots of transgenic tobacco plants, highlighting the role of these cis-elements in keeping the A9 promoter repressed in the roots of tobacco.

Deciphering the regulation of transporters and mitogen-activated protein kinase in arsenic and iron exposed rice.

This study investigates the influence of arsenic (As) and iron (Fe) on the molecular aspects of rice plants. The mRNA-abundance of As (OsLsi, OsPHT, OsNRAMP1, OsABCC1) and Fe (OsIRT, OsNRAMP1, OsYSL, OsFRDL1, OsVIT2, OsSAMS1, OsNAS, OsNAAT1, OsDMAS1, OsTOM1, OsFER) related genes has been observed in 12-d old As and Fe impacted rice varieties. Analyses of phytosiderophores synthesis and Fe-uptake genes affirm the existence of specialized Fe-uptake strategies in rice with varieties PB-1 and Varsha favouring strategy I and II, respectively. Expression of OsNAS3, OsVIT2, OsFER and OsABCC1 indicated PB-1's tolerance towards Fe and As. Analysis of mitogen-activated protein kinase cascade members (OsMKK3, OsMKK4, OsMKK6, OsMPK3, OsMPK4, OsMPK7, and OsMPK14) revealed their importance in the fine adjustment of As/Fe in the rice system. A conditional network map was generated based on the gene expression pattern that unfolded the differential dynamics of both rice varieties. The mating based split ubiquitin system determined the interaction of OsIRT1 with OsMPK3, and OsLsi1 with both OsMPK3 and OsMPK4. In-silico tools also confirmed the binding affinities of OsARM1 with OsLsi1, OsMPK3 and OsMPK4, and of OsIDEF1/OsIRO2 with OsIRT1 and OsMPK3, supporting our hypothesis that OsARM1, OsIDEF1, OsIRO2 were active in the connections discovered by mbSUS.

DNA N-glycosylases Ogg1 and EndoIII as components of base excision repair in Plasmodium falciparum organelles.

The integrity of genomes of the two crucial organelles of the malaria parasite - an apicoplast and mitochondrion in each cell - must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1leader-GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway.

MAP kinases may mediate regulation of the cell cycle in rice by E2F2 phosphorylation.

E2F is the key transcription factor that determines the proliferative status of cells by regulating the G1/S phase of the cell cycle. In this study, we show that in rice (Oryza sativa), OsE2F2 is a phosphorylation target of MAP kinases. The MAP kinases OsMPK3, OsMPK4, and OsMPK6 interact with and phosphorylate OsE2F2. Next, we determined the serine and threonine residues that could play a role in the phosphorylation of OsE2F2. Subsequently, our study suggests a possible link between MAP kinase-mediated OsE2F2 phosphorylation and its impact on DNA proliferation in the roots of rice seedlings. Finally, we found positive feedback regulation of OsMPK4 by OsE2F2. Therefore, our study hints at the potential impact of MAP kinase signaling on the cell cycle of rice plants.

Electro-organic green synthesis of dicyano-2-(2-oxoindolin-3-ylidene) malononitriles using molecular iodine as catalyst.

The first electrochemical molecular iodine promoted, domino reactions for the green synthesis of biologically relevant dicyano 2-(2-oxoindolin-3-ylidene) malononitriles (11 examples, up to 94% yield) from readily available isatin derivatives, malononitrile, and iodine at room temperature have been presented. This synthesis method showed tolerance towards various EDG and EWG and was completed in a short reaction time at the constant low current density of 5 mA cm-2 in the low redox potential range of -0.14 to 0.07 V. The present study exhibited by-product-free formation, easy operation, and product isolation. In particular the formation of a C[double bond, length as m-dash]C bond was observed at room temperature with a high atom economy. Furthermore, in the present study, the electrochemical behavior of dicyano 2-(2-oxoindolin-3-ylidene) malononitrile derivatives using a cyclic voltammetry (CV) technique in 0.1 M NaClO4 in acetonitrile solution was studied. All the chosen substituted isatin exhibited well-defined diffusion-controlled quasi-reversible redox peaks except 5-substituted derivatives. This synthesis could serve as an alternative strategy to synthesize other biologically important oxoindolin-3-ylidene malononitrile derivatives.

MKKK20 works as an upstream triple-kinase of MKK3-MPK6-MYC2 module in Arabidopsis seedling development.

The mitogen-activated protein kinase (MAPK) cascade is involved in several signal transduction processes in eukaryotes. Here, we report a mechanistic function of MAP kinase kinase kinase 20 (MKKK20) in light signal transduction pathways. We show that MKKK20 acts as a negative regulator of photomorphogenic growth at various wavelengths of light. MKKK20 not only regulates the expression of light signaling pathway regulatory genes but also gets regulated by the same pathway genes. The atmyc2 mkkk20 double mutant analysis shows that MYC2 works downstream to MKKK20 in the regulation of photomorphogenic growth. MYC2 directly binds to the promoter of MKKK20 to modulate its expression. The protein-protein interaction study indicates that MKKK20 physically interacts with MYC2, and this interaction likely suppresses the MYC2-mediated promotion of MKKK20 expression. Further, the protein phosphorylation studies demonstrate that MKKK20 works as the upstream kinase of MKK3-MPK6-MYC2 module in photomorphogenesis.

Regulation of photosynthetic light reaction proteins via reversible phosphorylation.

The regulation of photosynthesis occurs at different levels including the control of nuclear and plastid genes transcription, RNA processing and translation, protein translocation, assemblies and their post translational modifications. Out of all these, post translational modification enables rapid response of plants towards changing environmental conditions. Among all post-translational modifications, reversible phosphorylation is known to play a crucial role in the regulation of light reaction of photosynthesis. Although, phosphorylation of PS II subunits has been extensively studied but not much attention is given to other photosynthetic complexes such as PS I, Cytochrome b6f complex and ATP synthase. Phosphorylation reaction is known to protect photosynthetic apparatus in challenging environment conditions such as high light, elevated temperature, high salinity and drought. Recent studies have explored the role of photosynthetic protein phosphorylation in conferring plant immunity against the rice blast disease. The evolution of phosphorylation of different subunits of photosynthetic proteins occurred along with the evolution of plant lineage for their better adaptation to the changing environment conditions. In this review, we summarize the progress made in the research field of phosphorylation of photosynthetic proteins and highlights the missing links that need immediate attention.

Plasmodium Ape1 is a multifunctional enzyme in mitochondrial base excision repair and is required for efficient transition from liver to blood stage infection.

The malaria parasite has a single mitochondrion which carries multiple tandem repeats of its 6 kb genome encoding three proteins of the electron transport chain. There is little information about DNA repair mechanisms for mitochondrial genome maintenance in Plasmodium spp. Of the two AP-endonucleases of the BER pathway encoded in the parasite nuclear genome, the EndoIV homolog PfApn1 has been identified as a mitochondrial protein with restricted functions. We explored the targeting and biochemical properties of the ExoIII homolog PfApe1. PfApe1 localized in the mitochondrion and exhibited AP-site cleavage, 3'-5' exonuclease, 3'-phosphatase, nucleotide incision repair (NIR) and RNA cleavage activities indicating a wider functional role than PfApn1. The parasite enzyme differed from human APE1 in possessing a large, disordered N-terminal extension. Molecular modelling revealed conservation of structural domains but variations in DNA-interacting residues and an insertion in the α-8 loop suggested differences with APE1. Unlike APE1, where AP-site cleavage and NIR activities could be mutually exclusive based on pH and Mg2+ ion concentration, PfApe1 was optimally active under similar conditions suggesting that it can function both as an AP-endonuclease in BER and directly cleave damaged bases in NIR under similar physiological conditions. To investigate the role of Ape1 in malaria life cycle, we disrupted the gene by double-cross-over homologous recombination. Ape1 knockout (KO) P. berghei parasites showed normal development of blood and mosquito stages. However, inoculation of mice with Ape1 KO salivary gland sporozoites revealed a reduced capacity to initiate blood stage infection. Ape1 KO parasites underwent normal liver stage development until merozoites egressed from hepatocytes. Our results indicated that the delay in pre-patent period was due to the inability of Ape1 KO merosomes to infect erythrocytes efficiently.

Reciprocal silencing, transcriptional bias and functional divergence of homeologs in polyploid cotton (gossypium).

Polyploidy is an important force in the evolution of flowering plants. Genomic merger and doubling induce an extensive array of genomic effects, including immediate and long-term alterations in the expression of duplicate genes ("homeologs"). Here we employed a novel high-resolution, genome-specific, mass-spectrometry technology and a well-established phylogenetic framework to investigate relative expression levels of each homeolog for 63 gene pairs in 24 tissues in naturally occurring allopolyploid cotton (Gossypium L.), a synthetic allopolyploid of the same genomic composition, and models of the diploid progenitor species. Results from a total of 2177 successful expression assays permitted us to determine the extent of expression evolution accompanying genomic merger of divergent diploid parents, genome doubling, and genomic coevolution in a common nucleus subsequent to polyploid formation. We demonstrate that 40% of homeologs are transcriptionally biased in at least one stage of cotton development, that genome merger per se has a large effect on relative expression of homeologs, and that the majority of these alterations are caused by cis-regulatory divergence between the diploid progenitors. We describe the scope of transcriptional subfunctionalization and 15 cases of probable neofunctionalization among 8 tissues. To our knowledge, this study represents the first characterization of transcriptional neofunctionalization in an allopolyploid. These results provide a novel temporal perspective on expression evolution of duplicate genomes and add to our understanding of the importance of polyploidy in plants.

Pelvic hydatidosis mimicking a malignant multicystic ovarian tumor.

Echinococcosis is a multisystem disease and has propensity to involve any organ, an unusual anatomical site, and can mimic any disease process. Primary peritoneal echinococcosis is known to occur secondary to hepatic involvement but occasional cases of primary peritoneal hydatid disease including pelvic involvement have also been reported. We report here 1 such case of primary pelvic hydatidosis mimicking a malignant multicystic ovarian tumor where there was no evidence of involvement of the liver or spleen. Our patient, a 27-year-old female, was detected to have a large right cystic adnexal mass on per vaginal examination which was confirmed by ultrasonography. Her biochemical parameters were normal and CA-125 levels, though mildly raised, were below the cut off point. She underwent surgery and on exploratory laparotomy, another cystic mass was found attached to the mesentery of the small gut. The resected cysts were processed histopathologically. On cut sections both large cysts revealed numerous daughter cysts. Microscopic examination of fluid from the cysts revealed free scolices with hooklets and the cyst wall had a typical laminated membrane with inner germinal layer containing degenerated protoplasmic mass. The diagnosis of pelvic hydatid disease was confirmed and patient was managed accordingly. Hydatid disease must be considered while making the differential diagnosis of pelvic cystic masses, especially in endemic areas.

A bHLH transcription factor, MYC2, imparts salt intolerance by regulating proline biosynthesis in Arabidopsis.

MYC2, a bHLH TF, acts as regulatory hub within several signaling pathways by integration of various endogenous and exogenous signals which shape plant growth and development. However, its involvement in salt stress regulation is still elusive. This study has deciphered a novel role of MYC2 in imparting salt stress intolerance by regulating delta1 -pyrroline-5-carboxylate synthase1 (P5CS1) gene and hence proline synthesis. P5CS1 is a rate-limiting enzyme in the biosynthesis of proline. Y-1-H and EMSA studies confirmed the binding of MYC2 with the 5'UTR region of P5CS1. Transcript and biochemical studies have revealed MYC2 as a negative regulator of proline biosynthesis. Proline is necessary for imparting tolerance toward abiotic stress; however, its overaccumulation is toxic for the plants. Hence, studying the regulation of proline biosynthesis is requisite to understand the mechanism of stress tolerance. We have also studied that MYC2 is regulated by mitogen-activated protein kinase (MAPK) cascade mitogen-activated protein kinase kinase 3-MPK6 and vice versa. Altogether, this study demonstrates salt stress-mediated activation of MYC2 by MAPK cascade, regulating proline biosynthesis and thus salt stress.

Transcriptional regulation of anther development in Arabidopsis.

This review focuses on the current knowledge of transcription factors involved in Arabidopsis anther development. Anther development is a multistage process and controlled by a complex network of transcription factors acting in spatio/temporal manner. Molecular understanding of anther developmental pathway is critical from the perspective of controlling male fertility and hybrid generation. Generation of hybrid lines relies upon the effective mechanisms of controlling the process of pollen development and pollen release. Controlling any developmental program requires a good knowledge of regulatory pathways governing that developmental program. In a regulatory pathway, transcription factors represent an important link between the developmental program and response of genes to growth regulators and environmental signals. Therefore, identifying the entire cohort of anther specific transcription factors is an essential step towards the molecular understanding of regulatory networks involved in pollen formation and pollen release.

Global analysis of gene expression in cotton fibers from wild and domesticated Gossypium barbadense.

Gossypium barbadense is widely cultivated because of its extra-long staple cotton with superior luster, silkiness and high yield. These economically important traits were selected during initial domestication of an agronomically inferior wild ancestor, followed by millennia of human-mediated selection. To reveal the effects of this history on the cotton fiber transcriptome, we conducted comparative expression profiling on mechanically isolated fiber cells at three different stages encompassing early, mid, and late fiber elongation in wild (K101) and domesticated (Pima S-7) accessions, using a microarray platform that interrogates 42,429 unigenes. The distribution of differentially expressed genes across developmental stages was different in the two accessions, with a shift toward greater change earlier in cultivated than in wild G. barbadense. Approximately 4200 genes were differentially expressed between wild and domesticated accessions at one or more of the stages studied. Domestication appears to have led to enhanced modulation of cellular redox levels and the avoidance or delay of stress-like processes. Prolonged fiber growth in cultivated relative to wild G. barbadense is associated with upregulation of signal transduction and hormone signaling genes and down-regulation of cell wall maturation genes. Clues are provided into the processes and genes that may unwittingly have been selected by humans during domestication and development of modern elite lines. Several of the transcriptomic differences between wild and domesticated G. barbadense described here appear to have parallels in a second domesticated cotton species, Gossypium hirsutum, suggesting that replicated domestication of two different species has resulted in overlapping, parallel, metabolic transformations.

Biometric Attendance and Big Data Analysis for Optimizing Work Processes.

Although biometric attendance management is available, large healthcare organizations have difficulty in big data analysis for optimization of work processes. The aim of this project was to assess the implementation of a biometric attendance system and its utility following big data analysis. In this prospective study the implementation of biometric system was evaluated over 3 month period at our institution. Software integration with other existing systems for data analysis was also evaluated. Implementation of the biometric system could be successfully done over a two month period with enrollment of 10,000 employees into the system. However generating reports and taking action this large number of staff was a challenge. For this purpose software was made for capturing the duty roster of each employee and integrating it with the biometric system and adding an SMS gateway. This helped in automating the process of sending SMSs to each employee who had not signed in. Standalone biometric systems have limited functionality in large organizations unless it is meshed with employee duty roster.

Evaluation of the Serum Levels of Nitric Oxide among Diabetic Patients and its Correlation with Lipid Profile as well as Oxidative Stress in North Indian Setting.

INTRODUCTION: Diabetes mellitus is a disease with a rapidly increasing prevalence, needs continue research for novel methods to both prevent and treat this disorder. Obesity and decreased physical activity are the major risk factor for the development of diabetes. Recently the emphasis is focused on oxidative stress in pathogenesis of this disease. AIM: To assess the serum levels of Nitric Oxide (NO) among diabetic patients and its correlation with lipid profile as well as oxidative stress in north Indian setting. MATERIALS AND METHODS: This was a cross-sectional study. Subjects suffering from type 2 diabetes for more than 1 year and age between 30 to 50 years with hyperuricaemia were included in the study. The patients were divided into three groups: Group I- Type 2 diabetics with dyslipidemia and hyperuricaemia, Group II- Type 2 diabetics with dyslipidemia and normouricaemia and Group III- Type 2 diabetics with normolipidemia and normouricaemia. RESULTS: The nitric oxide level was significantly lower in Group I and Group II than Group III. The oxidative stress parameters had poor correlation with NO level in all the groups. CONCLUSION: Our data suggests that there is definite role of Nitric Oxide (NO) in pathogenesis of type -2 diabetes mellitus with dyslipidemia and hyperuricaemia.