Genetic analyses of brassinosteroid control of flowering time in Arabidopsis thaliana

A main developmental switch in the life cycle of a flowering plant is the transition from vegetative to reproductive growth. To maximize reproductive success, the timing of the floral transition must be precisely controlled. In Arabidopsis, at least four genetic pathways, the photoperiod, the autonomous, the vernalization, and the gibberellin (GA) pathways, which integrate endogenous and environmental signals, regulate the timing of this transition. A genetic screen was performed to identify additional components in the flowering-regulating network. This resulted in the isolation of two alleles of bri1 as enhancers of the late-flowering phenotype of the autonomous mutant luminidependens (ld). BRI1 encodes an LRR-RLK (leucine-rich repeats receptor-like kinase) that functions as a receptor for brassinosteroids (BRs), thus the result of the screen indicated that BRI1 or BRs could play a role in the floral timing. The aim of this thesis was to define and compare the roles of BRI1 and BRs in floral transition. The studies were extended with examination of potential genetic interactions between BRs, GAs, and abscisic acid (ABA) in the control of the transition to the reproductive phase in Arabidopsis. To place BRI1 in the flowering-genetic network, genetic and molecular-genetic approaches were used. Based on the analyses of various double mutants, which included combinations between bri1 and known flowering-time mutants, it was concluded that the BRI1 pathway has only a limited interaction with the photoperiod and the gibberellin pathways, and functions independently from vernalization. BRI1 functions in part through the autonomous pathway. Synergistic interaction between the BRI1 and the autonomous pathways was further confirmed by gene-expression studies. The bri1 autonomous/FRI lines exhibited enhanced expression of the potent floral repressor FLC, which is known to be regulated by the autonomous and FRI pathways. The increased levels of FLC was accompanied by reduced expression of the downstream targets, FT, SOC1, LFY, whose expression is important for the floral transition to occur. Moreover, specific reduction of FLC via RNAi accelerated flowering of the bri1 ld double mutant. Based on the presented results, a model was proposed that describes the BRI1 role in flowering-time control, where BRI1 promotes flowering by genetically interacting with the autonomous pathway to repress the potent floral repressor FLC. To define the relationship between BRI1 and BRs, the flowering-time phenotypes of the bri1 mutant and a BR-biosynthesis mutant cpd were compared. It could be inferred from these studies that the BR pathway also interacts with the autonomous pathway, and the bri1 phenotype could be partly explained by the BR-deficiency. However, due to differences in the flowering behavior of cpd and bri1, it was proposed that BRI1 also exerts its function through unknown BRI1-specific factors. The plausible mechanisms explaining additional flowering phenotypes of the bri1 were discussed. The role of BRs in the control of flowering time was also studied in the context of its possible interactions with the GA- and ABA-regulated pathways. The analyses of flowering phenotypes of double mutant combination deficient in BRs, ABA, GA did not reveal strong genetic interactions. The expression studies of key flowering-time genes in single and double hormone mutant combinations supported the model where the balance in the levels of these three hormones is necessary for appropriate timing of floral transition. Furthermore, based on the mild phenotype of the BR/ABA-deficient mutants, and the flowering behaviors of transgenic lines that overexpress DWF4, NCED3, and GA5, leading to BR-, ABA- and GA- overproducing phenotypes, respectively, it has been concluded that GA has a limiting, and BR, ABA have the supporting function in the control of flowering time in Arabidopsis. Attempts were made to verify whether the BR-pathway converge on the promoter of LFY, which is one of the floral-pathways integrators. For this purpose, the LFY::LUC+ reporter system was constructed, validated, and tested for BR-induction. No clear activation of the LFY promoter in the BR-overproducing lines was observed. In summary, the results described here provide evidence that both the BR-receptor BRI1 and brassinosteroids are important factors of floral-regulating network in Arabidopsis. Surprisingly, however, BRI1 exerts its effects on flowering only partially through the BR-regulated pathway. The nature of BRI1-specific effects on flowering time remains to be investigated

Pain management and functional recovery after pericapsular nerve group (PENG) block for total hip arthroplasty: A prospective, randomized, double-blinded clinical trial

Background: The immediate postoperative period after total hip arthroplasty can be associated with significant pain. Therefore, this study aimed to evaluate the effect of pericapsular nerve block on pain management and functional recovery after total hip arthroplasty. Methods: This prospective, randomized, double-blinded, placebo-controlled trial was conducted on 489 adult patients scheduled for total hip arthroplasty, ASA 1-2, operated under spinal analgesia. Participants were assigned to receive either a pericapsular nerve group (PENG) block with 20 mL of 0.5% ropivacaine or a sham block. Results: The primary outcome measure was the postoperative NRS score in motion. The secondary outcomes were cumulative opioid consumption, the time to the first opioid, and functional recovery. Demographic characteristics were similar in both groups. Intraoperative pain scores were significantly lower in patients who received the PENG block than in the control group (p < 0.0001). Also, the time to the first opioid was considerably longer in the PENG group (p < 0.0001). Additionally, 24% of PENG patients did not require opioids (p < 0.0001). Conclusions: The pericapsular nerve group showed significantly decreased opioid consumption and improved functional recovery. Pericapsular nerve group block improved pain management and postoperative functional recovery following total hip arthroplasty

A systematic survey in Arabidopsis thaliana of transcription factors that modulate circadian parameters

<p>Abstract</p> <p>Background</p> <p>Plant circadian systems regulate various biological processes in harmony with daily environmental changes. In <it>Arabidopsis thaliana</it>, the underlying clock mechanism is comprised of multiple integrated transcriptional feedbacks, which collectively lead to global patterns of rhythmic gene expression. The transcriptional networks are essential within the clock itself and in its output pathway.</p> <p>Results</p> <p>Here, to expand understanding of transcriptional networks within and associated to the clock, we performed both an <it>in silico </it>analysis of transcript rhythmicity of transcription factor genes, and a pilot assessment of functional phenomics on the <it>MYB</it>, <it>bHLH</it>, and <it>bZIP </it>families. In our <it>in silico </it>analysis, we defined which members of these families express a circadian waveform of transcript abundance. Up to 20% of these families were over-represented as clock-controlled genes. To detect members that contribute to proper oscillator function, we systematically measured rhythmic growth <it>via </it>an imaging system in hundreds of misexpression lines targeting members of the transcription-factor families. Three transcription factors were found that conferred aberrant circadian rhythms when misexpressed: <it>MYB3R2</it>, <it>bHLH69</it>, and <it>bHLH92</it>.</p> <p>Conclusion</p> <p>Transcript abundance of many transcription factors in Arabidopsis oscillates in a circadian manner. Further, a developed pipeline assessed phenotypic contribution of a panel of transcriptional regulators in the circadian system.</p

The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples

Transcriptional shift and metabolic adaptations during Leishmania quiescence using stationary phase and drug pressure as models

Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages

Unveiling drug-tolerant and persister-like cells in Leishmania braziliensis lines derived from patients with cutaneous leishmaniasis

IntroductionResistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania.MethodsWe studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR.Results and discussionWe demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC50 after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&amp;D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis

Genome diversity of Leishmania aethiopica

Leishmania aethiopica is a zoonotic Old World parasite transmitted by Phlebotomine sand flies and causing cutaneous leishmaniasis in Ethiopia and Kenya. Despite a range of clinical manifestations and a high prevalence of treatment failure, L. aethiopica is one of the most neglected species of the Leishmania genus in terms of scientific attention. Here, we explored the genome diversity of L. aethiopica by analyzing the genomes of twenty isolates from Ethiopia. Phylogenomic analyses identified two strains as interspecific hybrids involving L. aethiopica as one parent and L. donovani and L. tropica respectively as the other parent. High levels of genome-wide heterozygosity suggest that these two hybrids are equivalent to F1 progeny that propagated mitotically since the initial hybridization event. Analyses of allelic read depths further revealed that the L. aethiopica - L. tropica hybrid was diploid and the L. aethiopica - L. donovani hybrid was triploid, as has been described for other interspecific Leishmania hybrids. When focusing on L. aethiopica, we show that this species is genetically highly diverse and consists of both asexually evolving strains and groups of recombining parasites. A remarkable observation is that some L. aethiopica strains showed an extensive loss of heterozygosity across large regions of the nuclear genome, which likely arose from gene conversion/mitotic recombination. Hence, our prospection of L. aethiopica genomics revealed new insights into the genomic consequences of both meiotic and mitotic recombination in Leishmania

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent.

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context

Genetic Analyses of Interactions among Gibberellin, Abscisic Acid, and Brassinosteroids in the Control of Flowering Time in Arabidopsis thaliana

Genetic interactions between phytohormones in the control of flowering time in Arabidopsis thaliana have not been extensively studied. Three phytohormones have been individually connected to the floral-timing program. The inductive function of gibberellins (GAs) is the most documented. Abscisic acid (ABA) has been demonstrated to delay flowering. Finally, the promotive role of brassinosteroids (BRs) has been established. It has been reported that for many physiological processes, hormone pathways interact to ensure an appropriate biological response.We tested possible genetic interactions between GA-, ABA-, and BR-dependent pathways in the control of the transition to flowering. For this, single and double mutants deficient in the biosynthesis of GAs, ABA, and BRs were used to assess the effect of hormone deficiency on the timing of floral transition. Also, plants that over-express genes encoding rate-limiting enzymes in each biosynthetic pathway were generated and the flowering time of these lines was investigated.Loss-of-function studies revealed a complex relationship between GAs and ABA, and between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs. Gain-of-function studies revealed that GAs were clearly limiting in their sufficiency of action, whereas increases in BRs and ABA led to a more modest phenotypic effect on floral timing. We conclude from our genetic tests that the effects of GA, ABA, and BR on timing of floral induction are only in partially coordinated action

The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples