SCALiR: A Web Application for Automating Absolute Quantification of Mass Spectrometry-Based Metabolomics Data.

Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming an important approach for studying complex biological systems but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process that is often performed manually. Currently, there are few options for open-source software tools that can automatically calculate metabolite concentrations. Herein, we introduce SCALiR (standard curve application for determining linear ranges), a new web-based software tool specifically built for this task, which allows users to automatically transform LC-MS signals into absolute quantitative data (https://www.lewisresearchgroup.org/software). SCALiR uses an algorithm that automatically finds the equation of the line of best fit for each standard curve and uses this equation to calculate compound concentrations from the LC-MS signal. Using a standard mix containing 77 metabolites, we show a close correlation between the concentrations calculated by SCALiR and the expected concentrations of each compound (R2 = 0.99 for a y = x curve fitting). Moreover, we demonstrate that SCALiR reproducibly calculates concentrations of midrange standards across ten analytical batches (average coefficient of variation 0.091). SCALiR can be used to calculate metabolite concentrations either using external calibration curves or by using internal standards to correct for matrix effects. This open-source and vendor agnostic software offers users several advantages in that (1) it requires only 10 s of analysis time to compute concentrations of >75 compounds, (2) it facilitates automation of quantitative workflows, and (3) it performs deterministic evaluations of compound quantification limits. SCALiR therefore provides the metabolomics community with a simple and rapid tool that enables rigorous and reproducible quantitative metabolomics studies.

The Pseudomonas syringae pv. tomato DC3000 effector HopD1 interferes with cellular dynamics associated with the function of the plant immune protein AtNHR2B.

The plant pathogenic bacterium Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plant Arabidopsis thaliana, and conditionally in Nicotiana benthamiana. The pathogenicity of Pst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. The Pst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging of Nicotiana benthamiana plants transiently co-expressing HopD1 fused to the epitope haemagglutinin (HopD1-HA) with AtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.

Structure and regulation of full-length human leucine-rich repeat kinase 1.

The human leucine-rich repeat kinases (LRRKs), LRRK1 and LRRK2 are large and unusually complex multi-domain kinases, which regulate fundamental cellular processes and have been implicated in human disease. Structures of LRRK2 have recently been determined, but the structure and molecular mechanisms regulating the activity of the LRRK1 as well as differences in the regulation of LRRK1 and LRRK2 remain unclear. Here, we report a cryo-EM structure of the LRRK1 monomer and a lower-resolution cryo-EM map of the LRRK1 dimer. The monomer structure, in which the kinase is in an inactive conformation, reveals key interdomain interfaces that control kinase activity as we validate experimentally. Both the LRRK1 monomer and dimer are structurally distinct compared to LRRK2. Overall, our results provide structural insights into the activation of the human LRRKs, which advance our understanding of their physiological and pathological roles.

Predicting a Stable Dimeric Form of the PD1-PDL1 Complex: Implications for Understanding the PD1 Activation Mechanism.

The activation of T cells is typically accompanied by inhibitory mechanisms within which the programmed cell death (PD1) receptor stands out. Upon binding the ligands PDL1 and PDL2, PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling is activated upon the ligands' binding remains an open question. Several experimental facts suggest that the activation of the PD1-PLD1 pathway depends on the interaction with an unknown partner at the cellular membrane. In this work, we investigate the possibility that the target of PD1-PDL1 is the same PD1-PDL1 complex. We combined molecular docking with molecular dynamics and umbrella sampling simulations to explore different binding modes and assess the complexes' stability. We predicted a stable dimeric form of the extracellular domains of the PD1-PDL1 complex. This dimeric complex has an affinity comparable to the PD1-PDL1 interaction and resembles the form of a linear lattice. We proposed a new model for PD1 activation where the PD1-PDL1 dimeric form could facilitate the interaction of the intracellular domains of PD1 and the further binding and activation of the SHP2 phosphatase. This model might explain the inhibitory effect of anti-PD1/PDL1 antibodies through the prevention of the formation of the PD1-PDL1 dimers and, subsequently, the abrogation of the SHP2 phosphatase activation.

Emotional dysregulation in women with endometriosis with cyclical and non-cyclical chronic pelvic pain.

BACKGROUND: Endometriosis is a pathophysiological condition characterized by glands and stroma outside the uterus in regions such as the bladder, ureter, fallopian tubes, peritoneum, ovaries, and even in extra pelvic sites. One of the main clinical problems of endometriosis is chronic pelvic pain (CPP), which considerably affects the patients' quality of life. Patients with endometriosis may, cyclically or non-cyclically (80% of cases) experience CPP. High levels of anxiety and depression have been described in patients with endometriosis related to CPP; however, this has not been evaluated in endometriosis women with different types of CPP. Therefore, the research question of this study was whether there is a difference in the emotional dysregulation due to the type of pain experienced by women with endometriosis? METHODS: This work was performed in the National Institute of Perinatology (INPer) in Mexico City from January 2019 to March 2020 and aimed to determine if there are differences in emotional dysregulation in patients with cyclical and non-cyclical CPP. 49 women from 18 to 52 years-old diagnosed with endometriosis presenting cyclical and non-cyclical CPP answered several batteries made up of Mini-Mental State Examination, Visual Analog Scale, Beck's Depression Inventory, State Trait-Anxiety Inventory, and Generalized Anxiety Inventory. Mann-Whitney U and Student's t-test for independent samples to compare the difference between groups was used. Relative risk estimation was performed to determine the association between non-cyclical and cyclical CPP with probability of presenting emotional dysregulation. RESULTS: We observed that patients with non-cyclical CPP exhibited higher levels of depression and anxiety (trait-state and generalized anxiety) than patients with cyclical pain, p < 0.05 was considered significant. No differences were observed in pain intensity, but there was a higher probability of developing emotional dysregulation (anxiety or depression) in patients with non-cyclical CPP. No differences were observed in cognitive impairment. CONCLUSIONS: Our data suggest that patients with non-cyclical (persistent) CPP present a higher emotional dysregulation than those with cyclical pain.

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Transmission Dynamics and Effectiveness of Control Measures during COVID-19 Surge, Taiwan, April-August 2021.

An unprecedented surge of COVID-19 cases in Taiwan in May 2021 led the government to implement strict nationwide control measures beginning May 15. During the surge, the government was able to bring the epidemic under control without a complete lockdown despite the cumulative case count reaching >14,400 and >780 deaths. We investigated the effectiveness of the public health and social measures instituted by the Taiwan government by quantifying the change in the effective reproduction number, which is a summary measure of the ability of the pathogen to spread through the population. The control measures that were instituted reduced the effective reproduction number from 2.0-3.3 to 0.6-0.7. This decrease was correlated with changes in mobility patterns in Taiwan, demonstrating that public compliance, active case finding, and contact tracing were effective measures in preventing further spread of the disease.

The endoplasmic reticulum contributes to lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.

Emergence potential of monkeypox in the Western Pacific Region, July 2022.

Although new cases of monkeypox have been expected in the Western Pacific Region (WPR) since the virus emerged in Europe earlier this year, there have been only a few reported cases across the WPR (New Zealand 2, Singapore 6, South Korea 1, Taiwan 2), other than a limited number of cases (compared to numbers of cases seen elsewhere in the world) in Australia (33), as of July 15, 2022. In our short communication, we highlight two key reasons for this: i) international travel has still not fully resumed in the WPR following the COVID-19 pandemic, and ii) local public health measures to counter the spread of COVID-19 have not been completely relaxed. We provide supporting evidence for both of these reasons.

Methods for Quantifying the Metabolic Boundary Fluxes of Cell Cultures in Large Cohorts by High-Resolution Hydrophilic Liquid Chromatography Mass Spectrometry.

Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from bloodstream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment.

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway.

Unraveling a Conserved Conformation of the FG Loop upon the Binding of Natural Ligands to the Human and Murine PD1.

The activation of T cells is normally accompanied by inhibitory mechanisms within which the PD1 receptor stands out. PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions upon binding the ligands PDL1 and PDL2. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling pathway is activated upon the ligands' binding remains an open question. In this work, we used molecular dynamics simulations to assess the differences of the PD1 motion in the free state and in complex with the ligands. We found that, in both human and murine systems, the binding of PDL1 and PDL2 stabilizes the conformation of the FG loop similarly. This result, combined with the conservation of the FG loop residues across species, suggests that the conformation of the FG loop is somehow related to the signaling process. We also found a high similarity between the PD1-PDL1 structures with the variable region of an antibody structure, where the FG loop occupies a similar position to the CDR3 light chain.

LRRK2 recruitment, activity, and function in organelles.

Protein coding mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Infarto de miocardio con arterias coronarias no obstruidas en pediatría: una afección poco conocida / Myocardial infarction with unobstructed coronary arteries in pediatrics: a little-known entity

Resumen El infarto de miocardio con arterias coronarias no obstruidas (MINOCA, por sus siglas en inglés) ha ganado importancia en los últimos 20 años gracias a la dilucidación de etiologías fisiopatológicas diferentes de las causas obstructivas del flujo coronario. Diversos estudios han evidenciado una prevalencia variable, la cual es más alta en las mujeres. Se han descrito dos grupos de alteraciones en la reactividad coronaria que afectan el flujo: las causas epicárdicas y las causas microvasculares. El diagnóstico de MINOCA es de exclusión; por lo tanto, inicialmente se deben descartar otras posibles causas de isquemia, como miocarditis, miocardiopatía séptica, choque hipovolémico por trauma o quemaduras, y enfermedades renales o pulmonares. Los reportes y estudios de esta enfermedad suelen incluir pacientes adultos o mayores de 18 años. Se presenta el caso de una paciente de 16 años con antecedente de tetralogía de Fallot corregida en etapa de lactante menor y reemplazo valvular pulmonar con bioprótesis y ampliación del tronco pulmonar a los 11 años, quien ingresó con dolor torácico de características coronarias. Cursó con un diagnóstico de MINOCA por exclusión en una institución de cuarto nivel en Cali, Colombia. El diagnóstico de MINOCA en edad pediátrica es raro; sin embargo, es importante saber acerca de su existencia para brindar a los pacientes el mejor manejo disponible, de manera que se aseguren los mejores desenlaces a largo plazo.

Abstract Myocardial infarction with non-obstructed coronary arteries (MINOCA) has gained importance in the last 20 years, due to the elucidation of physiopathological etiologies different from the obstructive causes of coronary flow. Different studies have shown variable prevalence, being higher in women. Different causes have been evidenced in the studies found in two groups of alterations in coronary reactivity: epicardial causes and microvascular causes. The diagnosis of MINOCA must be a diagnosis of exclusion. Therefore, other possible causes of ischemia, such as myocarditis, septic cardiomyopathy, hypovolemic shock due to trauma or burns, renal or pulmonary diseases, should be ruled out initially. The reports and studies done around this pathology usually include adult patients and people older than 18 years. We present the case of a 16-year-old patient with a history of Tetralogy of Fallot corrected as an infant and pulmonary valve replacement with bioprothesis and enlargement of the pulmonary trunk at 11 years of age who was admitted with chest pain of coronary characteristics. The diagnosis after multiples studies and exclusion of other causes was MINOCA in a fourth level institution in Cali, Colombia.

Differential Effects of IL2Rα and IL15Rα over the Stability of the Common Beta-Gamma Signaling Subunits of the IL2 and IL15 Receptors.

Interleukin (IL) 2 and IL15 are two members of the common gamma chain cytokine family, involved in the regulation of the T cell differentiation process. Both molecules use a specific alpha subunit, IL2Rα and IL15Rα, and share the same beta and gamma chains signaling receptors. The presence of the specific alpha subunit modulates the T cell ability to compete for both soluble cytokines while the beta and gamma subunits are responsible for the signal transduction. Recent experimental results point out that the specific alpha subunits modulate the capacity of IL2 and IL15 to induce the differentiation of stimulated T cells. In other membrane receptors, the outcome of the signal transduction has been associated with the strength of the interaction of the signaling subunits. Here, we investigate how IL2Rα and IL15Rα modulate the stability of their signaling complexes by combining molecular dynamics simulations and free energy calculations. Our simulations predict that IL2Rα binding destabilizes the ß-γc interaction mediated by IL2, while IL15Rα has the opposite effect. These results explain the ability of IL2Rα and IL15Rα to modulate the signaling outcome and suggest new strategies for the development of better CD8+ T cell differentiation protocols for adoptive cell transfer (ACT).

Exploring the conformational dynamics of PD1 in complex with different ligands: What we can learn for designing novel PD1 signaling blockers?

Activation of T cells triggers the expression of regulatory molecules like the programmed cell death 1 (PD1) protein. The association of PD1 with the natural ligands PDL1 and PDL2 induces an inhibitory signal that prevents T cells from proliferating and exerting effector functions. However, little is known about how the binding of the ligands induce the PD1 inhibitory signal over T cells effector functions. Here, we explore the dynamics of PD1 free, and in complex with different PDL1 variants as well as the therapeutic antibodies nivolumab and pembrolizumab in order to assess the conformational changes in PD1 related to the signaling process. Our simulations suggest a pre-conformational selection mechanism for the binding of the different PDL1 variants, while an induced-fit model fits better for the molecular recognition process of the therapeutic antibodies. A deep analysis of the changes on PD1 movement upon the binding to different ligands revealed that as larger is the difference in the conformation adopted by loop C'D with respect to the complex with PDL1 is higher the ligand ability to reduce the PD1 inhibitory signaling. This behavior suggests that targeting specific conformations of this loop can be useful for designing therapies able to recover T cells effector functions.