Arabidopsis Transcriptome Analysis Reveals Key Roles of Melatonin in Plant Defense Systems

Melatonin is a ubiquitous molecule and exists across kingdoms including plant species. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. Much less attention has been drawn to its affect on genome-wide gene expression. To comprehensively investigate the role(s) of melatonin at the genomics level, we utilized mRNA-seq technology to analyze Arabidopsis plants subjected to a 16-hour 100 pM (low) and 1 mM (high) melatonin treatment. The expression profiles were analyzed to identify differentially expressed genes. 100 pM melatonin treatment significantly affected the expression of only 81 genes with 51 down-regulated and 30 up-regulated. However, 1 mM melatonin significantly altered 1308 genes with 566 up-regulated and 742 down-regulated. Not all genes altered by low melatonin were affected by high melatonin, indicating different roles of melatonin in regulation of plant growth and development under low and high concentrations. Furthermore, a large number of genes altered by melatonin were involved in plant stress defense. Transcript levels for many stress receptors, kinases, and stress-associated calcium signals were up-regulated. The majority of transcription factors identified were also involved in plant stress defense. Additionally, most identified genes in ABA, ET, SA and JA pathways were up-regulated, while genes pertaining to auxin responses and signaling, peroxidases, and those associated with cell wall synthesis and modifications were mostly down-regulated. Our results indicate critical roles of melatonin in plant defense against various environmental stresses, and provide a framework for functional analysis of genes in melatonin-mediated signaling pathways

Genotypic resistance testing improves antiretroviral treatment outcomes in a cohort of adolescents in Cameroon: Implications in the dolutegravir‑era

Background: Acquired drug resistance (ADR) is common among adolescents living with perinatal HIV (APHI) in sub-Saharan Africa (SSA). Personalized management has the potential to improve pediatric antiretroviral therapy (ART), even in the presence of long-term treatment and HIV-1 subtype diversity. Objective: We sought to evaluate the effect of HIV-1 mutational profiling on immuno-virological response and ADR among APHI. Methods: A cohort-study was conducted from 2018-2020 among 311 APHI receiving ART in Cameroon. Clinical, immunological and virological responses were measured at enrolment (T1), 6-months (T2) and 12-months (T3). Immunological failure (IF: CD4<250 cells/mm3), VF (viremia≥1000 copies/ml), and ADR were analyzed, with p<0.05 considered significant. Results: Mean age was 15(±3) years; male-female ratio was 1:1; median [IQR] ART-duration was 36[21-81] months. At T1, T2, and T3 respectively, adherence-level was 66.4%, 58.3% and 66.5%; 14 viral clades were found, driven by CRF02_AG (58.6%); ADR-mutations favored increased switch to second-line ART (16.1%, 31.2%, and 41.9%, p<0.0001). From T1-T3 respectively, there were declining rates of IF (25.5%, 18.9%, and 9.83, p<0.0001), VF (39.7%, 39.9%, and 28.2%, p=0.007), and HIVDR (96.4%, 91.7%, and 85.0%, p=0.099). Predictors of ADR were being on first-line ART (p=0.045), high viremia at enrolment (AOR=12.56, p=0.059), and IF (AOR=5.86, p=0.010). Of note, optimized ART guided by mutational profile (AOR=0.05, p=0.002) was protective. Moreover, full Tenofovir+Lamivudine+Dolutegravir efficacy was predicted in 77% and 62% of APHI respectively after first- and second-line failure. Conclusions: Among APHI in this SSA setting, viral mutational profiling prompts the use of optimized Dolutegravir-based ART regimens, leading to improved immuno-virological response and declining ADR burdens. Thus, implementing personalized HIV medicine in this vulnerable population would substantially improve ART response and the achievement of the 95-95-95 goals in these underserved populations

A new method for the in situ generation of chiral and achiral vinylboranes, and their use as reactive dienophiles in Diels-Alder reactions

Vita.Achiral vinyldihaloboranes, trivinylborane were generated in situ via a newly developed methodology which involves the reaction of vinyltributyltin with BBr$sb3$ or BCl$sb3.$ These compounds proved to be highly activated dienophiles in Diels-Alder reactions with simple dienes. Kinetic studies revealed that vinyldibromoborane is the most reactive dienophile among them. With a bimolecular rate constant of $rm 2times 10sp{-3} Msp{-1}ssp{-1}$ for its reaction with isoprene it is only second to tetracyanoethylene in its reactivity. This tremendous reactivity is apparently due to electronic effects of the bromine atoms bonded to boron. The dienophiles studied: vinyldibromoborane, vinyldichloroborane, divinylbromoborane, and trivinylborane gave the normal regiochemical products as predicted by Frontier Molecular Orbital Theory with 1- and 2-substituted-1,3-butadienes. Ab initio calculations indicate that vinyldihaloboranes adopt a planar structure, therefore the observed regioselectivities are electronically controlled. The involvement of a 4 + 3 transition state has been predicted for these reactions and this is proposed to be responsible for the endo-selectivities obtained for theses systems. Good to outstanding regioselectivities and endo-selectivities were observed comparable to Diels-Alder reactions catalyzed by Lewis acids. Thus vinyldibromoborane reacted with 2-tert-butyl-1,3-butadiene in less than 15 min at 25$spcirc$C to afford 4-tert-butyl-3-cyclohexen-1-ol and 3-tert-butyl-3-cyclohexen-1-ol in a 94:6, after oxidation with alkaline hydrogen peroxide. The other dienophiles gave regioselectivities $>$90%. The yields were good to excellent. The reaction of vinyldibromoborane with 1-phenyl-1,3-butadiene resulted in $>$99% regio- and endo-selectivity, and therefore a synthetically useful reaction. Chiral vinylboranes with auxilliaries derived from the hydroboration of chiral alkenes such as limonene, longifolene, $beta$-pinene were also studied. A modest chiral induction of 41% ee was obtained for the endo product of the reaction between dilongifolylvinylborane and cyclopentadiene. The vinylborane with camphosulfonyl groups as the chiral auxillaries was synthesized easily. However, this dienophile proved to be very stable towards Diels-Alder reactions with cyclopentadiene at up to 55$spcirc$C

Effect of melatonin on paraquat-induced oxidative stress.

<p>Arabidopsis leaves were detached and floated in solution containing 0, 10(top row) or presence (bottom row) of 1 mM melatonin. After 48 hours, leaves exposed to paraquat in the absence of melatonin were photobleached while leaves incubated with melatonin during exposure to paraquat remained green.</p

qRT-PCR analysis confirming overall results of RNA-seq experiments.

<p>The fold changes in transcript levels identified in RNA-seq experiments for 15 selected genes are graphed in A (up-regulated) and C (down-regulated). qRT-PCR was performed using the same samples for RNA-seq experiments with primers for the selected genes showing up-regulated (B) or down-regulated (D) by 1 mM melatonin. All q-RT-PCR were repeated four times. * p<0.05, ** p<0.01, ***p<0.001.</p

Schematic overview of genes exhibiting a change of 2-fold or more in response to melatonin associated with stress response and signaling.

<p>Fold change in transcript levels is represented by the color scale with darkest blue indicating genes with the greatest increase in transcript levels (4-fold or greater) and darkest red indicating genes with the greatest decrease in transcript levels (at least 4-fold). Each gene involved in stress responses is represented once.</p

Gene Ontology classification into molecular function of all genes significantly (p<0.05) differentially expressed in response to 1 mM and 100 pM melatonin.

<p>Genes were classified into at least one category using AmiGO GO slimmer. The proportion of genes that fall into each category were calculated by dividing the number of genes assigned to a category by the total number of genes that could be classified for each treatment.</p><p>*Indicates Molecular function is unknown.</p

Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.

<p>Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.</p

Venn Diagram representing the overlap of the molecular responses to low (100 pM) and high (1 mM) melatonin treatments.

<p>The number in the parenthesis indicates the number of genes altered by melatonin for each treatment.</p