The structure of Leptospira interrogans GAPDH sheds light into an immunoevasion factor that can target the anaphylatoxin C5a of innate immunity

Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans. This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host’s innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms. Besides, we have characterized the enzyme’s kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other α-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components

Background Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. Methods We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. Key Results We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a. In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. ConclusionXAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components.This work constitutes a partial fulfilment of the Graduate Program ‘Doctorado en Ciencias Biomédicas’ of the Universidad Nacional Autónoma de México in which Karla V. García-Cruz developed this project. We acknowledge the Consejo Nacional de Ciencia y Tecnología (CONACYT, México) that provided her scholarship. This work was supported by CONACYT: 240180 and 180380; PAPIIT, UNAM: IN203214-3; IN203113-3; IN203814-3; IN211516 and BFU2012-34821 from MINECO (Spain) to C.G., and an institutional grant from Fundación Ramón Areces to Centro de Biología Molecular Severo Ochoa. We thank David Cruz Sánchez and Diana Romo for technical and logistical support, respectively.Peer reviewe

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Crystal structure and SAXS analysis of the immune evasive factor GAPDH from Leptospira interrogans

1 p.Background: Immune evasive factors from pathogenic bacteriainclude metabolic enzymes that fulfill housekeeping roles intra-cellularly but which can contribute to infectivity when released as part of the bacterial secretome. The glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been characterized as a moonlighting protein with immune evasive roles in many gram-negative and gram-positive bacteria (1-2). GAPDH from several bacterial pathogens are known to interfere with the C5a-C5aR1 signaling pathway by binding to C5a. There-fore, research to determine the structural features enabling C5a sequestration and the potential for pharmacological intervention represent interesting alternatives to fight bacterial infections.Methods: The gene for Leptospira interrogans (Li)GAPDH wasamplified from gDNA and subcloned into a bacterial expression vector. Expression and purification of LiGAPDH was performed following established procedures. The structure of LiGAPDH wasdetermined by X-ray crystallography and the shape in solution calculated from small-angle X-ray diffraction (SAXS). A crosslinkingassay with BMOE helped to demonstrate the association between LiGAPDH and human C5a. GAPDH activity was measured with standard enzymatic assays and inhibition by two natural products (curcumin and anacardic acid) was evaluated.Results: We have cloned, expressed and purified GAPDH from Leptospira interrogans. We have crystallized it and solved its struc-ture by X-ray crystallography. In addition, we have restored itsshape in solution, validating the crystallographic model. LiGAPDH appears to be able to engage in a transient complex with human C5a, which we have trapped by crosslinking with BMOE, a highlyspecific reagent that crosslinks thiols < 6-8˚A away. We have analyzed the inhibitory potency and inhibition modality of two natural products with antimicrobial properties: anacardic acid and curcumin. Of them, only curcumin showed inhibition potential.Conclusions: The structure of the immune evasive factor GAPDH from Leptospira interrogans has been determined and is now available for experimental and computational drug discovery campaigns. Although weakly, LiGAPDH can interact with human C5a, suggesting that avidity effects caused by the concentration of GAPDH on the bacterium’s cell wall could enhance the C5a sequestration capacity of L. interrogans. Furthermore, curcumin has emerged as an interesting broad-spectrum inhibitor of LiGAPDH.Peer reviewe

A Dynamic Gene Regulatory Network Model That Recovers the Cyclic Behavior of Arabidopsis thaliana Cell Cycle.

Cell cycle control is fundamental in eukaryotic development. Several modeling efforts have been used to integrate the complex network of interacting molecular components involved in cell cycle dynamics. In this paper, we aimed at recovering the regulatory logic upstream of previously known components of cell cycle control, with the aim of understanding the mechanisms underlying the emergence of the cyclic behavior of such components. We focus on Arabidopsis thaliana, but given that many components of cell cycle regulation are conserved among eukaryotes, when experimental data for this system was not available, we considered experimental results from yeast and animal systems. We are proposing a Boolean gene regulatory network (GRN) that converges into only one robust limit cycle attractor that closely resembles the cyclic behavior of the key cell-cycle molecular components and other regulators considered here. We validate the model by comparing our in silico configurations with data from loss- and gain-of-function mutants, where the endocyclic behavior also was recovered. Additionally, we approximate a continuous model and recovered the temporal periodic expression profiles of the cell-cycle molecular components involved, thus suggesting that the single limit cycle attractor recovered with the Boolean model is not an artifact of its discrete and synchronous nature, but rather an emergent consequence of the inherent characteristics of the regulatory logic proposed here. This dynamical model, hence provides a novel theoretical framework to address cell cycle regulation in plants, and it can also be used to propose novel predictions regarding cell cycle regulation in other eukaryotes

The structure of Leptospira interrogans GAPDH sheds light into an immunoevasion factor that can target the anaphylatoxin C5a of innate immunity

15 p.-7 fig.-4 tab.Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans.This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host’s innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms.Besides, we have characterized the enzyme’s kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other a-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.This work was funded by Spanish Ministerio de Ciencia, Innovación y Universidades-FEDER grants RTI2018-102242-B-I00 (to MCV) and PID2019-104912RB-I00 (to SRdC); and the Spanish Ministerio de Ciencia e Innovación-Recovery, Transformation and Resilience Plan (PRTR) grant PDC2022-133713-I00. It was also funded by Grants S2017/BMD-3673 and S2022/BMD-7278 of the Regional Government of Madrid and the European Commission – NextGenerationEU through CSIC’s Global Health Platform (“PTI Salud Global”) (SGL2103020) (to SRdC and MCV), and the CSIC Special Intramural Grant PIE-201620E064 (to MCV). It was additionally supported by the Research Network on Complement in Health and Disease (RED2022-134750-T). SRdC was also supported by the CIBER de Enfermedades Raras. KdlP was supported by an Industrial PhD grant (IND2018-010094) awarded by the Spanish Ministerio de Economía y Competitividad.Peer reviewe

Attractor robustness analysis.

<p>Random networks with similar structure to <i>A. thaliana</i> CC GRN were less tolerant to perturbations than original CC GRN. The frequency of perturbations that recovered the original attractor after a perturbation in the Boolean functions, is shown in: (A), where the red line indicates that <i>A. thaliana</i> CC GRN recovers its original attractor in 68% of perturbations (the median of random networks was 18.55% and mean 19.12% ± 13.86 SD). When transitions between network configurations are perturbed (B), <i>A. thaliana</i> CC GRN recovers its original attractor in 88% (vertical red line) of perturbations, while the median of random networks that recover the original attractor was 24.2% (mean 24.6% ± 18.2 SD). Vertical blue line indicates the 95% quantile. 1000 random networks were analyzed.</p

Attractors recovered by simulations of loss- or gain-of-function mutants of four CC components.

<p>(A) The simulation of loss of CDKB1;1 function produced only one cyclic attractor with period 7 that resembles G1 → S → G2 → G1 cycle, whereas in (B) with simulation of loss of KRP1 function, one cyclic attractor was attained, which has period 11 and comprises 100% of the initial conditions. This attractor is almost identical to WT phenotype but without KRP1. With the simulation of APC/C gain-of-function, a single attractor with period 7 was recovered, which is shown in (C) and is consistent with an endoreduplication cycle. Attractors obtained with the simulation of E2Fa overexpression are shown in (D). Two attractors were found, one of them has period 10 and the 40.48% of the initial conditions converge to that cycle that is closely similar to the WT CC attractor. The second attractor that correspond to E2Fa overexpression has period 8 and it is very similar to the endoreduplication attractor of loss of CDKB1;1 function, which comprises 59.52% of possible network configurations.</p

Phenotypes of gain-of-function mutations in CC components.

<p>Summary of mutant phenotypes and recovered attractors simulating that mutation. <b>A</b> means that the result of simulation is in Agreement with the data available in the reference(s). <b>PA</b> means it is Partially Agrees with evidence, because not all expected traits were reproduced by the attractor but this does not contradict the mutant phenotype. <b>NR</b> are attractors that do not make sense with expected behavior and therefore, the model did Not Recover the mutant phenotype.</p><p>Phenotypes of gain-of-function mutations in CC components.</p