The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Source Tracing of Leishmania donovani in Emerging Foci of Visceral Leishmaniasis, Western Nepal.

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance.

Four layer multi-omics reveals molecular responses to aneuploidy in Leishmania.

Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond.

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani.

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

Genome instability drives epistatic adaptation in the human pathogen Leishmania.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

Effectiveness of the Bilateral and Bilevel Erector Spinae Plane Block (ESPB) in Pediatric Idiopathic Scoliosis Surgery: A Randomized, Double-Blinded, Controlled Trial.

BACKGROUND: This study aimed to compare the effect of the ultrasound-guided bilateral and bilevel erector spinae plane block (ESPB) on pain scores, opioid requirement, intraoperative motor-evoked potentials (MEPs), and stress response to surgery expressed by the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) versus standard analgesia methods following idiopathic scoliosis surgery. METHODS: This was a prospective, double-blinded, randomized controlled trial. Sixty patients aged 10 to 18 years and physical status ASA 1 or 2 were randomized into 2 equal groups, each receiving an ESPB or sham block. The primary outcome was the pain scores (Numerical Rating Scale, NRS) within 48 hours after spinal correction and fusion surgery for idiopathic thoracic scoliosis. The secondary outcomes were total opioid consumption, NLR, and PLR levels at 12 and 24 hours postoperatively and intraoperative MEPs. RESULTS: ESPB patients presented lower NRS scores, signifying less pain, at all time points (30, 60, 90, 120 min; and 6, 12, 24, and 48 h after surgery), all P <0.0001. The total opioid consumption, the incidence of nausea or vomiting, and the need for remifentanil and propofol during surgery were significantly lower in the ESPB group. The surgery-induced stress response expressed by NLR and PLR was considerably lower in the ESPB group. ESPB did not affect the intraoperative MEP's amplitude. CONCLUSIONS: ESPB is effective for postoperative analgesia, can reduce opioid consumption in patients undergoing scoliosis surgery, and reduces the stress response to surgery. ESPB does not interfere with neuromonitoring. LEVEL OF EVIDENCE: Level I.

Signal integration in the control of shoot branching.

Shoot branching is a highly plastic developmental process in which axillary buds are formed in the axil of each leaf and may subsequently be activated to give branches. Three classes of plant hormones, auxins, cytokinins and strigolactones (or strigolactone derivatives) are central to the control of bud activation. These hormones move throughout the plant forming a network of systemic signals. The past decade brought great progress in understanding the mechanisms of shoot branching control. Biological and computational studies have led to the proposal of two models, the auxin transport canalization-based model and the second messenger model, which provide mechanistic explanations for apical dominance.

The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) levels following erector spinae plane block (ESPB) in posterior lumbar decompression: a randomized, controlled trial.

PURPOSE: Neutrophile-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are the inflammatory biomarkers of the stress response. In this study, we aimed to evaluate the effects of erector spinae plane block (ESPB) on posterior lumbar decompression and stabilization by comparing NLR, PLR, postoperative pain, opioid consumption, and functional recovery between sham block and ESPB. METHODS: This was a prospective, double-blinded, randomized controlled trial in a tertiary referral hospital. Sixty patients were randomized into two equal groups, each receiving either a sham block or ESPB. The primary outcome was the NLR and PLR 12 h and 24 h after lumbar posterior decompression and stabilization. The secondary outcomes were total opioid consumption and pain score 24 h postoperatively. Also, functional recovery determined by getting out of bed, verticalization, and walking by the balcony were reviewed as secondary outcomes. RESULTS: Significant differences existed between the sham block and ESPB group in NLR (29.08 ± 12.29 vs. 16.97 ± 10.38; p < 0.0001) and PLR (556.77 ± 110.32 vs. 346.43 ± 117.34; p < 0.0001) 12 h after surgery. Also, there was a significant difference in NLR (p = 0.0466) and PLR (p < 0.0001) 24 h after surgery. In addition, there was a substantial difference in pain score, total opioid consumption, and functional recovery. CONCLUSION: ESPB performance during spinal surgery lowers NRL and PLR ratios 12 h and 24 h after surgery. In addition, ESPB provides better analgesia and improves functional recovery compared to sham block following posterior lumbar decompression and stabilization.

Bilateral Bi-Level Erector Spinae Plane Blocks as a Part of Opioid-Sparing Multimodal Analgesia in Scoliosis Surgery: A Case Series of Six Pediatric Patients.

Background and Aim: Postoperative pain after scoliosis surgery is severe and usually requires long-term intravenous opioid therapy. Local anesthetic options, such as wound infiltration, are limited and include neuraxial analgesia. However, they are rarely used due to side effects and inconsistent efficacy. We report an opioid-sparing multimodal analgesia regimen with bilateral erector spinae plane blocks. This case series evaluated the analgesic effect of the bilateral bi-level erector spinae plane blocks (ESP) in congenital and neurogenic scoliosis surgery. Patients and Methods: Six pediatric patients with congenital or neurogenic scoliosis underwent posterior spinal fusion involving 5 to 12 vertebral levels. Bilateral single-injection ESPB was performed at one or two levels before incision. Preoperatively, patients received intravenous dexamethasone. General anesthesia with endotracheal intubation and volume-controlled ventilation was performed via TIVA with remifentanil and propofol. During and after the procedure, the basic hemodynamic parameters, opioid consumption, pain scores (numerical rating scale/NRS), and possible block complications were monitored. Results: All the patients experienced minimal postoperative pain levels. In addition, on the first day after surgery, they had low opioid requirements with no side effects. Conclusions: ESPB in patients undergoing congenital and neurogenic scoliosis correction surgery seems to be an essential analgesic technique that may reduce both severities of pain and opioid consumption.

The Systems Architecture of Molecular Memory in Poplar after Abiotic Stress.

Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar (Populus× canescens) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across five major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression profiles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis (Arabidopsis thaliana) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-specific components contributes to the coordination of post-recovery responses to stress in woody plants.

Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana.

Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development. We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle. Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large-leaf mutants. Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general.

Facing the Future: Effects of Short-Term Climate Extremes on Isoprene-Emitting and Nonemitting Poplar.

Isoprene emissions from poplar (Populus spp.) plantations can influence atmospheric chemistry and regional climate. These emissions respond strongly to temperature, [CO2], and drought, but the superimposed effect of these three climate change factors are, for the most part, unknown. Performing predicted climate change scenario simulations (periodic and chronic heat and drought spells [HDSs] applied under elevated [CO2]), we analyzed volatile organic compound emissions, photosynthetic performance, leaf growth, and overall carbon (C) gain of poplar genotypes emitting (IE) and nonemitting (NE) isoprene. We aimed (1) to evaluate the proposed beneficial effect of isoprene emission on plant stress mitigation and recovery capacity and (2) to estimate the cumulative net C gain under the projected future climate. During HDSs, the chloroplastidic electron transport rate of NE plants became impaired, while IE plants maintained high values similar to unstressed controls. During recovery from HDS episodes, IE plants reached higher daily net CO2 assimilation rates compared with NE genotypes. Irrespective of the genotype, plants undergoing chronic HDSs showed the lowest cumulative C gain. Under control conditions simulating ambient [CO2], the C gain was lower in the IE plants than in the NE plants. In summary, the data on the overall C gain and plant growth suggest that the beneficial function of isoprene emission in poplar might be of minor importance to mitigate predicted short-term climate extremes under elevated [CO2]. Moreover, we demonstrate that an analysis of the canopy-scale dynamics of isoprene emission and photosynthetic performance under multiple stresses is essential to understand the overall performance under proposed future conditions.

Grassland species differentially regulate proline concentrations under future climate conditions: an integrated biochemical and modelling approach.

Proline (Pro) is a versatile metabolite playing a role in the protection of plants against environmental stresses. To gain a deeper understanding of the regulation of Pro metabolism under predicted future climate conditions, including drought stress, elevated temperature and CO2 , we combined measurements in contrasting grassland species (two grasses and two legumes) at multiple organisational levels, that is, metabolite concentrations, enzyme activities and gene expression. Drought stress (D) activates Pro biosynthesis and represses its catabolism, and elevated temperature (DT) further elevated its content. Elevated CO2 attenuated the DT effect on Pro accumulation. Computational pathway control analysis allowed a mechanistic understanding of the regulatory changes in Pro metabolism. This analysis indicates that the experimentally observed coregulation of multiple enzymes is more effective in modulating Pro concentrations than regulation of a single step. Pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) play a central role in grasses (Lolium perenne, Poa pratensis), and arginase (ARG), ornithine aminotransferase (OAT) and P5CR play a central role in legumes (Medicago lupulina, Lotus corniculatus). Different strategies in the regulation of Pro concentrations under stress conditions were observed. In grasses the glutamate pathway is activated predominantly, and in the legumes the ornithine pathway, possibly related to differences in N-nutritional status.

Physiological, biochemical, and genome-wide transcriptional analysis reveals that elevated CO2 mitigates the impact of combined heat wave and drought stress in Arabidopsis thaliana at multiple organizational levels.

Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level analysis (i.e. eco-physiological, biochemical, and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under ambient and elevated CO2 . The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2 O2 production, and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect.

A systematic review and meta-analysis of systematic and topical tranexamic acid administration in aesthetic plastic surgery.

INTRODUCTION: Tranexamic acid has been widely used in plastic surgery. However, its efficacy has yet to be fully established. This meta-analysis aimed to determine its effectiveness in aesthetic plastic surgery. METHODS: Following PRISMA guidelines, we conducted a meta-analysis of prospective randomised clinical trials that compared the effects of topical or systematic administration of tranexamic acid versus the control group in aesthetic plastic surgeries. The study was registered on the International Register of Systematic Reviews (PROSPERO) and is available online ( www.crd.york.uk/prospero , CRD42023492585). RESULTS: Eleven studies encompassing 960 patients were included for the synthesis after critical evaluation. Systematic (MD - 18.05, 95% Cl, - 22.01, - 14.09, p < 0.00001) and topical (MD - 74.93, 95% Cl, - 88.79, - 61.07, p < 0.00001) administration of tranexamic acid reduced total blood loss. Topical tranexamic acid reduced drainage output (p < 0.0006). CONCLUSION: Tranexamic acid reduced blood loss in aesthetic plastic surgery. More strictly defined RCTs, using high-quality methodology, are needed to evaluate the advantages and disadvantages of tranexamic acid in aesthetic plastic surgery.

Morphine (10, 20 mg) in a Postoperative Dressing Used with Patients After Surgical Debridement of Burn Wounds: A Prospective, Double-Blinded, Randomized Controlled Trial.

Objective: This is the first clinical trial to evaluate the analgesic effect of 10 and 20 mg of morphine used in a postoperative dressing with patients after surgical debridement of burn wounds. Approach: In this randomized controlled trial, 20 adult patients with third-degree flame burns, who had undergone surgical debridement under general anesthesia, were randomly assigned to either group A, whose members were treated with a burn dressing that contained 10 mg of morphine, or group B, whose members were treated with a burn dressing that contained 20 mg of morphine; the dressing was also soaked with octenidine and phenoxyethanol in the case of both groups. The plasma morphine concentrations were measured 1, 2, 3, and 6 h after surgery, while the level of pain intensity was determined on the Numeric Pain Rating Scale (NRS), and the occurrence of side effects was observed. Results: The serum morphine concentration levels were very low, but statistically different between the two groups at all time points. The NRS value was similar in both groups at all time points (p > 0.05). Despite this, in group B, the NRS value was 0 in all patients in postoperative hours 1, 2, and 3. No adverse effect of morphine sulfate was observed in any patient. Innovation: This project is the first clinical study to have demonstrated that morphine administered in dressings in concentrations of 0.02-0.08 mg/mL significantly reduces the occurrence of pain. Conclusion: The use of morphine in dressings after surgical treatment of burn wounds is very effective when it comes to pain management and is safe for the patient.

Analgesic Efficacy of Oxycodone in Postoperative Dressings after Surgical Treatment of Burn Wounds: A Randomised Controlled Trial.

INTRODUCTION: This study aimed to assess the analgesic efficacy of oxycodone at doses of 10 mg and 20 mg in dressings after surgery of burn wounds. MATERIAL AND METHODS: Twenty adult patients who underwent surgical treatment of third-degree burn wounds under general anaesthesia were included. Burn wounds were treated with dressings, to which oxycodone was added at 20 mg in Group 1 and 10 mg in Group 2. After the surgery, plasma oxycodone and noroxycodone concentrations were assayed, and pain intensity was assessed with Numerical Rating Scale (NRS). RESULTS: In Group 1, no patient reported pain; in Group 2, four patients reported pain. The pain intensity, according to NRS, was 1-8. Plasma concentration of oxycodone in the blood serum was in the range of 1.24-3.15 ng/mL and 1.09-1.28 ng/mL in Group 1 and Group 2, respectively. Noroxycodone was not detected in the plasma. Adverse effects were not observed in any of the treated patients. CONCLUSIONS: Oxycodone in dressings provides patients with adequate and safe analgesia.

Persistent T cell unresponsiveness associated with chronic visceral leishmaniasis in HIV-coinfected patients.

A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8+/CD8- T cells, in addition to a lower frequency of IFN-γ+ TIGIT- CD8+/CD8- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4+ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.