Sensitive, High-Throughput HLA-I and HLA-II Immunopeptidomics Using Parallel Accumulation-Serial Fragmentation Mass Spectrometry.

Comprehensive and in-depth identification of the human leukocyte antigen class I (HLA-I) and class II (HLA-II) tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is a powerful technology for direct identification of HLA peptides from patient-derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare and clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high-throughput, sensitive, and single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight MS on the Bruker timsTOF single-cell proteomics system (SCP). We demonstrate greater than twofold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation, and reduces input requirements to as few as 1e6 A375 cells for >800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen and noncanonical proteins. We also apply our optimized single-shot SCP acquisition methods to tumor-derived samples, enabling sensitive, high-throughput, and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry.

Comprehensive, in-depth identification of the human leukocyte antigen HLA-I and HLA-II tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is powerful technology for direct identification of HLA peptides from patient derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare, clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high throughput, sensitive, single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight mass spectrometry on the Bruker timsTOF SCP. We demonstrate >2-fold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation and reduces input requirements to as few as 1e6 A375 cells for > 800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen, and novel/unannotated open reading frames. We also apply our optimized single-shot SCP acquisition methods to tumor derived samples, enabling sensitive, high throughput and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

Calcineurin dephosphorylates topoisomerase IIß and regulates the formation of neuronal-activity-induced DNA breaks.

Neuronal activity induces topoisomerase IIß (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

Indirect effects of the SARS CoV-2 pandemic on the prevalence of breastfeeding: Modeling its impact. / Efectos indirectos de la pandemia por SARS CoV-2 en la prevalencia de lactancia materna: modelación de su impacto

INTRODUCTION: Breastfeeding has a protective effect against acute respiratory and diarrheal infections. There are psychological and social effects due to physical isolation in the population in the mother-child group. OBJECTIVE: To assess the impact on infant mortality due to a decrease in the prevalence of breastfeeding during 2020 due to the physical isolation against the SARS CoV-2 (COVID-19) pandemic in Colombia. MATERIALS AND METHODS: We used the population attributable risk approach taking into account the prevalence of breastfeeding and its potential decrease associated with the measures of physical isolation and the relative risk (RR) of the association between exclusive breastfeeding and the occurrence of acute infection consequences in the growth (weight for height) of children under the age of five through a mathematical modeling program. RESULTS: We found an increase of 11.39% in the number of cases of growth arrest in the age group of 6 to 11 months with a 50% decrease in breastfeeding prevalence, as well as an increase in the number of diarrhea cases in children between 1 and 5 months of age from 5% (5.67%) on, and an increased number of deaths in children under 5 years (9.04%) with a 50% decrease in the prevalence of exclusive breastfeeding. CONCLUSIONS: A lower prevalence of breastfeeding has an impact on infant morbidity and mortality in the short and medium-term. As a public health policy, current maternal and childcare strategies must be kept in order to reduce risks in the pediatric population.

Introducción. La lactancia materna tiene un efecto protector frente a infecciones respiratorias y diarreicas agudas. Hay efectos psicológicos y sociales por el aislamiento físico en la población en el grupo materno-infantil. Objetivo. Evaluar el eventual impacto en la mortalidad infantil de la disminución en la prevalencia de la lactancia materna durante el 2020 a causa del aislamiento físico por la pandemia del SARS CoV-2 (COVID-19) en Colombia. Materiales y métodos. Se utilizó el enfoque de riesgo atribuible poblacional, teniendo en cuenta la prevalencia de la lactancia materna y su potencial disminución asociada con las medidas de aislamiento físico y el riesgo relativo (RR) de la asociación entre la lactancia materna exclusiva y el efecto de la aparición de infecciones agudas en el crecimiento (peso para la altura) de niños menores de cinco años mediante un programa de modelamiento matemático. Resultados. Se registró un aumento del número casos de detención del crecimiento en el grupo etario de 6 a 11 meses de 11,39 % al disminuir en 50 % la prevalencia de la lactancia materna, así como un mayor número de casos por diarrea en los cinco primeros meses a partir del 5 % (5,67 %), y un incremento en el número de muertes en menores de 5 años (9,04 %) al disminuirse en 50 % la prevalencia de la lactancia materna. Conclusiones. Se registró un impacto en la morbilidad y la mortalidad infantil a corto y mediano plazo al disminuir la prevalencia en la lactancia materna. Como política pública en salud, deben mantenerse las estrategias actuales de atención materno-infantil para disminuir riesgos en la población infantil.

Indirect effects of the SARS CoV-2 pandemic on the prevalence of breastfeeding: Modeling its impact / Efectos indirectos de la pandemia por SARS CoV-2 en la prevalencia de lactancia materna: modelación de su impacto

Abstract | Introduction: Breastfeeding has a protective effect against acute respiratory and diarrheal infections. There are psychological and social effects due to physical isolation in the population in the mother-child group. Objective: To assess the impact on infant mortality due to a decrease in the prevalence of breastfeeding during 2020 due to the physical isolation against the SARS CoV-2 (COVID-19) pandemic in Colombia. Materials and methods: We used the population attributable risk approach taking into account the prevalence of breastfeeding and its potential decrease associated with the measures of physical isolation and the relative risk (RR) of the association between exclusive breastfeeding and the occurrence of acute infection consequences in the growth (weight for height) of children under the age of five through a mathematical modeling program. Results: We found an increase of 11.39% in the number of cases of growth arrest in the age group of 6 to 11 months with a 50% decrease in breastfeeding prevalence, as well as an increase in the number of diarrhea cases in children between 1 and 5 months of age from 5% (5.67%) on, and an increased number of deaths in children under 5 years (9.04%) with a 50% decrease in the prevalence of exclusive breastfeeding. Conclusions: A lower prevalence of breastfeeding has an impact on infant morbidity and mortality in the short and medium-term. As a public health policy, current maternal and childcare strategies must be kept in order to reduce risks in the pediatric population.

Resumen | Introducción. La lactancia materna tiene un efecto protector frente a infecciones respiratorias y diarreicas agudas. Hay efectos psicológicos y sociales por el aislamiento físico en la población en el grupo materno-infantil. Objetivo. Evaluar el eventual impacto en la mortalidad infantil de la disminución en la prevalencia de la lactancia materna durante el 2020 a causa del aislamiento físico por la pandemia del SARS CoV-2 (COVID-19) en Colombia. Materiales y métodos. Se utilizó el enfoque de riesgo atribuible poblacional, teniendo en cuenta la prevalencia de la lactancia materna y su potencial disminución asociada con las medidas de aislamiento físico y el riesgo relativo (RR) de la asociación entre la lactancia materna exclusiva y el efecto de la aparición de infecciones agudas en el crecimiento (peso para la altura) de niños menores de cinco años mediante un programa de modelamiento matemático. Resultados. Se registró un aumento del número casos de detención del crecimiento en el grupo etario de 6 a 11 meses de 11,39 % al disminuir en 50 % la prevalencia de la lactancia materna, así como un mayor número de casos por diarrea en los cinco primeros meses a partir del 5 % (5,67 %), y un incremento en el número de muertes en menores de 5 años (9,04 %) al disminuirse en 50 % la prevalencia de la lactancia materna. Conclusiones. Se registró un impacto en la morbilidad y la mortalidad infantil a corto y mediano plazo al disminuir la prevalencia en la lactancia materna. Como política pública en salud, deben mantenerse las estrategias actuales de atención materno-infantil para disminuir riesgos en la población infantil.

Proteomic profiling dataset of chemical perturbations in multiple biological backgrounds.

While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of "connectivity" to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics .

Causal interactions from proteomic profiles: Molecular data meet pathway knowledge.

We present a computational method to infer causal mechanisms in cell biology by analyzing changes in high-throughput proteomic profiles on the background of prior knowledge captured in biochemical reaction knowledge bases. The method mimics a biologist's traditional approach of explaining changes in data using prior knowledge but does this at the scale of hundreds of thousands of reactions. This is a specific example of how to automate scientific reasoning processes and illustrates the power of mapping from experimental data to prior knowledge via logic programming. The identified mechanisms can explain how experimental and physiological perturbations, propagating in a network of reactions, affect cellular responses and their phenotypic consequences. Causal pathway analysis is a powerful and flexible discovery tool for a wide range of cellular profiling data types and biological questions. The automated causation inference tool, as well as the source code, are freely available at http://causalpath.org.

Avant-garde: an automated data-driven DIA data curation tool.

Several challenges remain in data-independent acquisition (DIA) data analysis, such as to confidently identify peptides, define integration boundaries, remove interferences, and control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. We present Avant-garde as a tool to refine DIA (and parallel reaction monitoring) data. Avant-garde uses a novel data-driven scoring strategy: signals are refined by learning from the dataset itself, using all measurements in all samples to achieve the best optimization. We evaluate the performance of Avant-garde using benchmark DIA datasets and show that it can determine the quantitative suitability of a peptide peak, and reach the same levels of selectivity, accuracy, and reproducibility as manual validation. Avant-garde is complementary to existing DIA analysis engines and aims to establish a strong foundation for subsequent analysis of quantitative mass spectrometry data.

The Impact of Rehabilitation-oriented Virtual Reality Device in Patients With Ischemic Stroke in the Early Subacute Recovery Phase: Study Protocol for a Phase III, Single-Blinded, Randomized, Controlled Clinical Trial.

BACKGROUND AND RATIONALE: Stroke is considered the most common cause of adult disability. Intensive rehabilitation protocols outperform nonintensive counterparts. The subacute stroke phase represents a potential window to recovery. Virtual reality (VR) has been shown to provide a more stimulating environment, allowing for increased patient compliance. However, the quality of current literature comparing VR with standard therapies is limited. Our aim is to measure the impact of VR versus standard therapy on the recovery of the upper limb motor function in patients with stroke in the early subacute recovery phase. METHOD: This is a randomized, controlled trial that will assign 262 patients to tailor-made standard rehabilitation (TMSR) or TMSR plus immersive VR device. The trial will be conducted in an urban rehabilitation clinic in the United States with expertise in the management of poststroke patients. Patients will be 18 to 70 years of age and in the early subacute period (30-90 days post ischemic stroke). The primary outcome will be the change of Fugl-Meyer Assessment-Upper Extremity (FMA-UE) score, measured at baseline and 13 weeks after randomization. The secondary outcome will be the change in the UK Functional Independence Measure and Functional Assessment Measure (UK FIM-FAM) score at the same time points. DISCUSSION: If the use of VR in the rehabilitation of patients with stroke proves to have a significant impact on their motor recovery, it will constitute an extremely important step into decreasing the functional impairment associated with stroke and the related health care expense burden.

Multi-OMICS analyses unveil STAT1 as a potential modifier gene in mevalonate kinase deficiency.

OBJECTIVES: The objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease. METHODS: Where single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings. RESULTS: This multiomics approach led to the identification of a single gene-STAT1-which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling. CONCLUSIONS: This study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.

Specter: linear deconvolution for targeted analysis of data-independent acquisition mass spectrometry proteomics.

Mass spectrometry with data-independent acquisition (DIA) is a promising method to improve the comprehensiveness and reproducibility of targeted and discovery proteomics, in theory by systematically measuring all peptide precursors in a biological sample. However, the analytical challenges involved in discriminating between peptides with similar sequences in convoluted spectra have limited its applicability in important cases, such as the detection of single-nucleotide polymorphisms (SNPs) and alternative site localizations in phosphoproteomics data. We report Specter (https://github.com/rpeckner-broad/Specter), an open-source software tool that uses linear algebra to deconvolute DIA mixture spectra directly through comparison to a spectral library, thus circumventing the problems associated with typical fragment-correlation-based approaches. We validate the sensitivity of Specter and its performance relative to that of other methods, and show that Specter is able to successfully analyze cases involving highly similar peptides that are typically challenging for DIA analysis methods.

Doublet N-Terminal Oriented Proteomics for N-Terminomics and Proteolytic Processing Identification.

The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.

MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state.

Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.

N-terminome analysis of the human mitochondrial proteome.

The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).

Agenesia del sacro / Sacro agenesis

Dentro del espectro de displasia caudal o síndrome de regresión caudal se encuentra la agenesia del sacro. El espectro de malformaciones de la masa celular caudal incluye las lumbosacras, las anorrectales y las urogenitales, así como anormalidades de miembros inferiores. Su presentación puede incluir una o más de las siguientes: Fusión de los miembros inferiores (sirenomielia), Agenesia lumbosacra, Atresia anal, Genitales anormales, Aplasia renal, Hipoplasia pulmonar. Puede asociarse con el síndrome de Potter. Su incidencia está aumentada en hijos de madres diabéticas. Las anormalidades espinales pueden ir desde la agenesia parcial del sacro hasta la agenesia total de las vértebras lumbosacras