Proteomic snapshot of saliva samples predicts new pathways implicated in SARS-CoV-2 pathogenesis.

BACKGROUND: Information on the microbiome's human pathways and active members that can affect SARS-CoV-2 susceptibility and pathogenesis in the salivary proteome is very scarce. Here, we studied a unique collection of samples harvested from April to June 2020 from unvaccinated patients. METHODS: We compared 10 infected and hospitalized patients with severe (n = 5) and moderate (n = 5) coronavirus disease (COVID-19) with 10 uninfected individuals, including non-COVID-19 but susceptible individuals (n = 5) and non-COVID-19 and nonsusceptible healthcare workers with repeated high-risk exposures (n = 5). RESULTS: By performing high-throughput proteomic profiling in saliva samples, we detected 226 unique differentially expressed (DE) human proteins between groups (q-value ≤ 0.05) out of 3376 unambiguously identified proteins (false discovery rate ≤ 1%). Major differences were observed between the non-COVID-19 and nonsusceptible groups. Bioinformatics analysis of DE proteins revealed human proteomic signatures related to inflammatory responses, central cellular processes, and antiviral activity associated with the saliva of SARS-CoV-2-infected patients (p-value ≤ 0.0004). Discriminatory biomarker signatures from human saliva include cystatins, protective molecules present in the oral cavity, calprotectins, involved in cell cycle progression, and histones, related to nucleosome functions. The expression levels of two human proteins related to protein transport in the cytoplasm, DYNC1 (p-value, 0.0021) and MAPRE1 (p-value, 0.047), correlated with angiotensin-converting enzyme 2 (ACE2) plasma activity. Finally, the proteomes of microorganisms present in the saliva samples showed 4 main microbial functional features related to ribosome functioning that were overrepresented in the infected group. CONCLUSION: Our study explores potential candidates involved in pathways implicated in SARS-CoV-2 susceptibility, although further studies in larger cohorts will be necessary.

Discrimination of Etiologically Different Cholestasis by Modeling Proteomics Datasets.

Cholestasis is characterized by disrupted bile flow from the liver to the small intestine. Although etiologically different cholestasis displays similar symptoms, diverse factors can contribute to the progression of the disease and determine the appropriate therapeutic option. Therefore, stratifying cholestatic patients is essential for the development of tailor-made treatment strategies. Here, we have analyzed the liver proteome from cholestatic patients of different etiology. In total, 7161 proteins were identified and quantified, of which 263 were differentially expressed between control and cholestasis groups. These differential proteins point to deregulated cellular processes that explain part of the molecular framework of cholestasis progression. However, the clustering of different cholestasis types was limited. Therefore, a machine learning pipeline was designed to identify a panel of 20 differential proteins that segregate different cholestasis groups with high accuracy and sensitivity. In summary, proteomics combined with machine learning algorithms provides valuable insights into the molecular mechanisms of cholestasis progression and a panel of proteins to discriminate across different types of cholestasis. This strategy may prove useful in developing precision medicine approaches for patient care.

Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study.

BACKGROUND AND AIMS: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol. METHODS: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot. RESULTS: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet. CONCLUSIONS: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids.

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease that affects between 1/50,000 and 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, and then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids (BAs) are kept in phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free BAs that result in liver damage. PFIC3 onset usually occurs at early ages, progresses rapidly, and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short-aged patients. To gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. We identified 6246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprogramming, cytoskeleton and extracellular matrix remodeling, and cell proliferation, were identified as the main drivers of the disease. Our results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients.

Construcción de una escala para medir relaciones de poder en el aula / Building a scale to measure power relations in the classroom

Se muestran los resultados de un estudio prospectivo, de una cohorte, de validación psicométrica que tuvo como objetivo validar un banco de 20 reactivos que buscaban medir las relaciones de poder en el aula. Estos reactivos se sometieron a juicio de expertos, a análisis de validez factorial y de invarianza de parámetros. Con los mejores reactivos, se armó la Escala de Relaciones de Poder en el Aula (ERPA), aplicada a una muestra de 198 estudiantes en el ámbito nacional. Los resultados indicaron que la ERPA presenta propiedades psicométricas que aseguran la calidad y precisión en sus mediciones. Este estudio permitió discutir las implicaciones teóricas y prácticas en la evaluación y fomento del aprendizaje universitario.

This paper presents the results of a prospective study of a cohort of psychometric validation aimed to validate a bank of 20 items that sought to measure the power relations in the classroom. These reactive were subjected to expert judgment, to analysis of factorial validity and invariance of parameters. With the best reagents, Power Relations Scale in the Classroom (PRSC). This scale was applied to a sample of 198 nationally students. The results indicated that the PRSC has psychometric properties that ensure the quality and accuracy in their measurements. This study allowed discussing theoretical and practical implications in the evaluation and promotion of college learning.

Relaciones estructurales entre estrategias de afrontamiento y síndrome de Burnout en personal de salud: un estudio de validez externa y de constructo / Structural relations between coping strategies and Burnout syndrome on health workers: A study of external and construct validity

El objetivo del estudio fue validar relaciones estructurales y funcionales hipotetizadas entre estrategias de afrontamiento y factores asociados al síndrome de burnout en muestras independientes de trabajadores de la salud, de diferentes hospitales y contextos de atención hospitalaria. Se aplicó el Maslach Burnout Inventory y la escala de Afrontamiento ante Riesgos Extremos adaptados para población mexicana; se analizaron las respuestas de 354 trabajadores de la salud de un hospital de tercer nivel y 300 de un hospital de referencia. Las muestras fueron intencionales, no probabilísticas, por cuotas. Los grupos se conformaron con personal de enfermería, para-médicos, servicios auxiliares y diagnóstico, médicos y médicos residentes. Los resultados confirmaron la estructura factorial de los instrumentos en las dos muestras; mostraron que el uso de estrategias activas de afrontamiento (control, búsqueda de información, soporte social, entre otras) tiene efectos protectores sobre los factores asociados al síndrome (cansancio emocional, despersonalización y realización personal en el trabajo), independientemente de la muestra, del contexto y del tipo de atención hospitalaria. Se discuten los resultados y sus implicaciones en relación con la validez externa y hallazgos en otras investigaciones relacionadas.

The aim of this study was to validate hypothesized structural and functional relationships between coping strategies and factors associated with burnout syndrome in independent samples of health workers from different hospitals and hospital care settings. We applied the Maslach Burnout Inventory and the scale of Coping with Extreme Risks adapted to the Mexican population, we analyzed the responses of354 health workers in a tertiary hospital and 300 from a reference hospital. The samples were intentional, non-probability, quota. The group was formed by nurses, paramedics, diagnostics services, physicians and resident physicians. Results confirmed the factor structure of the instruments in the two samples and showed that the use of active coping strategies (control, information seeking, social support, among others) has protective effects on the factors associated with the syndrome (emotional exhaustion, depersonalization and personal fulfillment at work), regardless of the sample, context and type of hospital care. We discuss the results and their implications in relation to external validity and findings in other related research.

Prevalencia delssíndrome de burnout y estrategis de afrontamiento durante una epidemia de influenz aah1n1 / Prevaence of burrnout syndrome and coping strategies uring an epidemmiology because of ah1n1 influenza

El objetivo de este estudio fue evaluar el síndrome de Burnout en una muestra de 477 trabajadores de la salud durante una epidemia por influenza AH1N1 en la Ciudad de México, e identificar si el uso de estrategias de afrontamiento tenía efectos protectores al Burnout. Se realizó un estudio descriptivo transversal con una muestra no probabilística por cuotas, utilizando como instrumentos el Maslach Burnout Inventory versión Human Services y la Escala de Afrontamiento Frente a Riesgo Extremos. Se hizo una distribución de frecuencia y de normalidad para analizar las diferencias, un análisis de varianza en Burnout y regresión lineal múltiple para estrategias de afrontamiento. A partir del análisis, se encontró que el 24% presentó el síndrome de Burnout en grado moderado (cansancio emocional y despersonalización, con presencia de realización personal); asimismo, fueron empleadas estrategias activas y pasivas. Se constató que el uso de estrategias de afrontamiento fue selectivo y las estrategias activas tuvieron una función moderadora al Burnout, protectora a la escala de cansancio emocional, que aumentó cuando presentaban realización personal. El estudio propone el desarrollo de investigaciones cuantitativas y cualitativas, así como el uso de modelos mixtos de intervención: centradas en las personas.

The aim of this study was to assess the Burnout syndrome in health workers during a contingency in Mexico City because of epidemiology AH1N1 influenza, and identify if the use of coping strategies had protective effects for the Burnout. We had a sample of 477 health workers and was performed a transversal descriptive study with a nonrandom quote sample. The instruments used were Maslach Burnout Inventory Human Services version and scale measuring coping face to extreme risks. It was performed a frequency and normality distribution to analyze the differences, and also an analysis of variance in Burnout, and multiple lineal regression for coping strategies. We found that 24% had Burnout (in emotional exhaustion and a moderate degree depersonalization, with the presence of personal accomplishment). They were also employed active and passive strategies. We found that the use of coping strategies was selective, active strategies had a moderating role to Burnout and protective to the scale of emotional exhaustion, which seems to increase when there is personal realization. We propose the development of quantitative and qualitative research and the use of mixed models of intervention: focusing on people.

Extravasación de orina espontánea por cálculo ureteral obstructivo: informe de dos casos y revisión de la literatura / Spontaneous urine tissular leak caused by ureteral calculus: report of two cases and literature review

Se presentan dos casos de extravasación espontánea de orina por rotura de la vía, secundaria a obstrucción ureteral distal por cálculo. El diagnóstico se estableció con urografía intravenosa, ultrasonografía y tomografía axil computadorizada. En ambos enfermos, la extracción del cálculo resolvió la extravasación. Se revisan etiopatogenia, cuadro clínico y diagnóstico diferencial. Está indicado el tratamiento de su etiología y la recuperación morfofuncional completa suele ser regla.