Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10.

Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Incidence of major cardiovascular events at 1, 2, and 5 years and mortality in incident patients on peritoneal dialysis regarding their nutritional status by bioimpedanciometry: A multicenter study in Colombia.

BACKGROUND: The nutritional status of incident patients on peritoneal dialysis (PD) has been associated with survival outcomes. Bioimpedanciometry (BCM) enables to establish a nutritional diagnosis, the volume status, and correlates these findings with survival. METHODS: This study used a retrospective multicenter historical cohort. RESULTS: In this study, which included 420 incident patients on peritoneal dialysis with a 5-year follow-up, a cumulative incidence of major adverse cardiovascular events (MACE) of 28.8% was found, being higher in the diabetic population at 36.8%. In regard to the nutritional status in this population, it was found that approximately 44% had altered nutritional status; 34% were found to be in sarcopenia; 6.7% sarcopenic obesity; and 2.8% in obesity (p < 0.001). In the survival analysis, a lower probability of survival was found in patients with overhydration (OH) greater than 3 L (p < 0.001) and in patients with altered nutritional status due to sarcopenia, sarcopenic obesity, and obesity (p 0.016). According to survival in the subgroup of the diabetic population, a lower probability of survival was found in this group of patients (p: 0.011). The overall mortality of the study population was 18%, being higher in the first 2 years, with the most important causes of mortality being cardiovascular. Of the deceased population, 51% were diabetic patients (p: 0.012). CONCLUSION: In incident patients on peritoneal dialysis, sarcopenic obesity, sarcopenia, overhydration status determined by BCM, and having a diagnosis of diabetes are related to a lower probability of survival; MACE outcomes are more frequent in the diabetic population.

Influence of non-osteoporotic treatments in patients on active anti-osteoporotic therapy: evidence from the OSTEOMED registry.

PURPOSE: To evaluate the effect of different non-osteoporotic drugs on the increase or decrease in the risk of incident fragility fractures (vertebral, humerus or hip) in a cohort of patients diagnosed with osteoporosis on active anti-osteoporotic therapy. METHODS: For this retrospective longitudinal study, baseline and follow-up data on prescribed non-osteoporotic treatments and the occurrence of vertebral, humerus or hip fractures in 993 patients from the OSTEOMED registry were analyzed using logistic regression models. The drugs evaluated with a possible beneficial effect were thiazides and statins, while the drugs evaluated with a possible harmful effect were antiandrogens, aromatase inhibitors, proton pump inhibitors, selective serotonin reuptake inhibitors, benzodiazepines, GnRH agonists, thyroid hormones, and oral and inhaled corticosteroids. RESULTS: Logistic regression analyses indicated that no treatment significantly improved fracture risk, with the only treatments that significantly worsened fracture risk being letrozole (OR = 0.18, p-value = 0.03) and oral corticosteroids at doses ≤ 5 mg/day (OR = 0.16, p-value = 0.03) and > 5 mg/day (OR = 0.27, p-value = 0.04). CONCLUSION: The potential beneficial or detrimental effects of the different drugs evaluated on fracture risk are masked by treatment with anabolic or antiresorptive drugs that have a more potent action on bone metabolism, with two exceptions: letrozole and oral corticosteroids. These findings may have important clinical implications, as patients receiving these treatments are not fully protected by bisphosphonates, which may imply the need for more potent anti-osteoporotic drugs such as denosumab or teriparatide.

Zika virus evolution and spread in the Americas.

Although the recent Zika virus (ZIKV) epidemic in the Americas and its link to birth defects have attracted a great deal of attention, much remains unknown about ZIKV disease epidemiology and ZIKV evolution, in part owing to a lack of genomic data. Here we address this gap in knowledge by using multiple sequencing approaches to generate 110 ZIKV genomes from clinical and mosquito samples from 10 countries and territories, greatly expanding the observed viral genetic diversity from this outbreak. We analysed the timing and patterns of introductions into distinct geographic regions; our phylogenetic evidence suggests rapid expansion of the outbreak in Brazil and multiple introductions of outbreak strains into Puerto Rico, Honduras, Colombia, other Caribbean islands, and the continental United States. We find that ZIKV circulated undetected in multiple regions for many months before the first locally transmitted cases were confirmed, highlighting the importance of surveillance of viral infections. We identify mutations with possible functional implications for ZIKV biology and pathogenesis, as well as those that might be relevant to the effectiveness of diagnostic tests.

Implementation of an integrated care strategy for child contacts of tuberculosis patients: a quasi-experimental study protocol.

BACKGROUND: Childhood tuberculosis continues to be a major public health problem. Although the visibility of the epidemic in this population group has increased, further research is needed. OBJECTIVE: To design, implement and evaluate an integrated care strategy for children under five years old who are household contacts of bacteriologically confirmed pulmonary tuberculosis patients in Medellín and the Metropolitan Area. METHODS: A quasi-experimental study in which approximately 300 children who are household contacts of bacteriologically confirmed pulmonary tuberculosis patients from Medellín and the Metropolitan Area will be evaluated and recruited over one year. A subgroup of these children, estimated at 85, who require treatment for latent tuberculosis, will receive an integrated care strategy that includes: some modifications of the current standardized scheme in Colombia, with rifampicin treatment daily for four months, follow-up under the project scheme with nursing personnel, general practitioners, specialists, professionals from other disciplines such as social work, psychology, and nutritionist. Additionally, transportation and food assistance will be provided to encourage treatment compliance. This strategy will be compared with isoniazid treatment received by a cohort of children between 2015 and 2018 following the standardized scheme in the country. The study was approved by the CIB Research Ethics Committee and UPB. CLINICALTRIALS: gov identifier NCT04331262. DISCUSSION: This study is expected to contribute to the development of integrated care strategies for the treatment of latent tuberculosis in children. The results will have a direct impact on the management of childhood tuberculosis contributing to achieving the goals proposed by the World Health Organization's End TB Strategy. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04331262 . Implementation of an Integrated Care Strategy for Children Contacts of Patients with Tuberculosis. Registered 2 April 2020.

Gender prediction based on University students' complex thinking competency: An analysis from machine learning approaches.

This article aims to study machine learning models to determine their performance in classifying students by gender based on their perception of complex thinking competency. Data were collected from a convenience sample of 605 students from a private university in Mexico with the eComplexity instrument. In this study, we consider the following data analyses: 1) predict students' gender based on their perception of complex thinking competency and sub-competencies from a 25 items questionnaire, 2) analyze models' performance during training and testing stages, and 3) study the models' prediction bias through a confusion matrix analysis. Our results confirm the hypothesis that the four machine learning models (Random Forest, Support Vector Machines, Multi-layer Perception, and One-Dimensional Convolutional Neural Network) can find sufficient differences in the eComplexity data to classify correctly up to 96.94% and 82.14% of the students' gender in the training and testing stage, respectively. The confusion matrix analysis revealed partiality in gender prediction among all machine learning models, even though we have applied an oversampling method to reduce the imbalance dataset. It showed that the most frequent error was to predict Male students as Female class. This paper provides empirical support for analyzing perception data through machine learning models in survey research. This work proposed a novel educational practice based on developing complex thinking competency and machine learning models to facilitate educational itineraries adapted to the training needs of each group to reduce social gaps existing due to gender.

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis.

BACKGROUND & AIMS: Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. METHODS: We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. RESULTS: MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and in vitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. CONCLUSIONS: Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. LAY SUMMARY: By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

Ecological relevance of non-perennial rivers for the conservation of terrestrial and aquatic communities.

River conservation efforts traditionally focus on perennial watercourses (i.e., those that do not dry) and their associated aquatic biodiversity. However, most of the global river network is not perennial and thus supports both aquatic and terrestrial biodiversity. We assessed the conservation value of nonperennial rivers and streams (NPRS) in one of Europe's driest regions based on aquatic (macroinvertebrates, diatoms) and terrestrial (riparian plants, birds, and carabid beetles) community data. We mapped the distribution of taxa at 90 locations and across wide environmental gradients. Using the systematic planning tool Marxan, we identified priority conservation sites under 2 scenarios: aquatic taxa alone or aquatic and terrestrial taxa together. We explored how environmental factors (runoff, flow intermittence, elevation, salinity, anthropogenic impact) influenced Marxan's site selection frequency. The NPRS were selected more frequently (over 13% on average) than perennial rivers when both aquatic and terrestrial taxa were considered, suggesting that NPRS have a high conservation value at the catchment scale. We detected an underrepresentation of terrestrial taxa (8.4-10.6% terrestrial vs. 0.5-1.1% aquatic taxa were unrepresented in most Marxan solutions) when priority sites were identified based exclusively on aquatic biodiversity, which points to a low surrogacy value of aquatic taxa for terrestrial taxa. Runoff explained site selection when focusing on aquatic taxa (all best-fitting models included runoff, r2  = 0.26-0.27), whereas elevation, salinity, and flow intermittence were more important when considering both groups. In both cases, site selection frequency declined as anthropogenic impact increased. Our results highlight the need to integrate terrestrial and aquatic communities when identifying priority areas for conservation in catchments with NPRS. This is key to overcoming drawbacks of traditional assessments based only on aquatic taxa and to ensure the conservation of NPRS, especially as NPRS become more prevalent worldwide due to climate change and increasing water demands.

Los esfuerzos de conservación fluvial se enfocan tradicionalmente en los cauces permanentes (aquellos que no se secan) y la biodiversidad acuática asociada. Sin embargo, la mayor parte de la red hidrográfica mundial no es permanente, por lo que sustenta biodiversidad tanto acuática como terrestre. Evaluamos el valor de conservación de los ríos y arroyos no permanentes (RANP) en una de las regiones más secas de Europa con datos de comunidades acuáticas (macroinvertebrados, diatomeas) y terrestres (escarabajos carábidos). Mapeamos la distribución de los taxones en 90 localidades que cubren gradientes ambientales amplios. Con la herramienta de planificación sistemática Marxan identificamos los sitios prioritarios de conservación bajo dos escenarios: considerando sólo los taxones acuáticos o los taxones acuáticos y terrestres juntos. Exploramos cómo los factores ambientales (escorrentía, intermitencia del caudal, altitud, salinidad, impacto antropogénico) influyeron sobre la frecuencia de selección de sitio de Marxan. Los RANP fueron seleccionados con mayor frecuencia (más del 13% en promedio) que los ríos permanentes cuando consideramos los taxones acuáticos y terrestres, lo que sugiere que los RANP tienen un valor elevado de conservación a escala de cuenca. Detectamos que los taxones terrestres estaban infrarrepresentados (8.4-10.6% taxones terrestres vs. 0.5-1.1% acuáticos no tuvieron representación en la mayoría de las soluciones de Marxan) cuando los sitios prioritarios para la conservación se identificaban exclusivamente con la biodiversidad acuática, lo que indica que los taxones acuáticos tienen un reducido valor indicador para los taxones terrestres. La escorrentía determinó la selección de sitios cuando se basó en los taxones acuáticos (los mejores modelos incluyeron la escorrentía, r2 = 0.26-0.27), mientras que la altitud, la salinidad y la intermitencia del caudal fueron más importantes cuando se consideraron ambos grupos. En ambos casos, la frecuencia de selección disminuyó conforme se incrementó el impacto antropogénico. Nuestros resultados resaltan la necesidad de integrar las comunidades terrestres y acuáticas a la identificación de las áreas prioritarias para la conservación de la biodiversidad en cuencas con RANP. Lo anterior es importante para superar las evaluaciones tradicionales basadas solamente en los taxones acuáticos y para garantizar la conservación de los RANP, especialmente ahora que estos son cada vez más frecuentes a nivel mundial debido al cambio climático y a la creciente demanda de agua.

Reproducibility of a protocol for standardized reading of chest X-rays of children household contact of patients with tuberculosis.

BACKGROUND: The interpretation of the chest radiograph may vary because it depends on the reader and due to the non-specificity of findings in tuberculosis (TB). We aim to assess the reproducibility of a standardized chest radiograph reading protocol in contacts of patients with pulmonary TB under the 5 years of age. METHODS: Descriptive, cross-sectional study with children under the age of five, household contacts of patients with confirmed pulmonary TB from Medellín, Bello and Itagüí (Colombia) between Jan-01-2015 and May-31-2016. Standardized reading protocol: two radiologists, blinded independent reading, use of template (Dr. Andronikou design) in case of disagreement a third reading was performed. Kappa coefficient for intra and inter observer agreement, and prevalence ratio were estimated of sociodemographic characteristics, TB exposure and interpretation of chest X-ray. RESULTS: From 278 children, standardized reading found 255 (91.7%) normal X-rays, 10 (3.6%) consistent with TB, and 13 (4.7%) other alterations. Global agreement was 91.3% (Kappa = 0.51). Inter-observer agreement between readers 1-2 was 90.0% (Kappa = 0.59) and 1-3 93.2% (Kappa = 0.59). Intra-observer agreement for reader 1 was 95.5% (Kappa = 0.86), 2 84.0% (Kappa = 0.51), and 3 94.7% (Kappa = 0.68). Greater inter-observer disagreement was between readers 1-2 for soft tissue density suggestive of adenopathy (4.6%), airspace opacification (1.17%) and pleural effusion (0.58%); between readers 1-3 for soft tissue density suggestive of adenopathy (4.2%), opacification of airspace (2.5%) and cavities (0.8%). CONCLUSIONS: Chest radiographs are an affordable tool that contributes to the diagnosis of TB, so having a standardized reading protocol showed good agreement and improves the reproducibility of radiograph interpretation.

Comorbidity and osteoporotic fracture: approach through predictive modeling techniques using the OSTEOMED registry.

PURPOSE: To examine the response to anti-osteoporotic treatment, considered as incident fragility fractures after a minimum follow-up of 1 year, according to sex, age, and number of comorbidities of the patients. METHODS: For this retrospective observational study, data from baseline and follow-up visits on the number of comorbidities, prescribed anti-osteoporotic treatment and vertebral, humerus or hip fractures in 993 patients from the OSTEOMED registry were analyzed using logistic regression and an artificial network model. RESULTS: Logistic regression showed that the probability of reducing fractures for each anti-osteoporotic treatment considered was independent of sex, age, and the number of comorbidities, increasing significantly only in males taking vitamin D (OR = 7.918), patients without comorbidities taking vitamin D (OR = 4.197) and patients with ≥ 3 comorbidities taking calcium (OR = 9.412). Logistic regression correctly classified 96% of patients (Hosmer-Lemeshow = 0.492) compared with the artificial neural network model, which correctly classified 95% of patients (AUC = 0.6). CONCLUSION: In general, sex, age and the number of comorbidities did not influence the likelihood that a given anti-osteoporotic treatment improved the risk of incident fragility fractures after 1 year, but this appeared to increase when patients had been treated with risedronate, strontium or teriparatide. The two models used classified patients similarly, but predicted differently in terms of the probability of improvement, with logistic regression being the better fit.

Absence of Long-Term Protection in Domestic Pigs Immunized with Attenuated African Swine Fever Virus Isolate OURT88/3 or BeninΔMGF Correlates with Increased Levels of Regulatory T Cells and Interleukin-10.

Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninΔMGF have previously been shown to induce high percentages of protection in domestic pigs against challenge with virulent virus. The results obtained in the present study show that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninΔMGF followed by a challenge with the virulent Benin 97/1 isolate at day 130 postimmunization did not trigger the mechanisms necessary to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of acute swine fever (ASF). Gamma interferon-producing cells peaked at day 24 postimmunization, declining thereafter. Surprisingly, the levels of regulatory T cells (Tregs) and interleukin-10 (IL-10) were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.IMPORTANCE The duration of immunity for any vaccine candidate is crucial. In the case of African swine fever virus vaccine candidates, this issue has received little attention. Attenuated viruses have proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, the duration of immunity and the immune responses induced over a duration of 130 days were studied during prechallenge and after challenge of pigs immunized with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate, BeninΔMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate or BeninΔMGF virus, animals were not protected against challenge with the virulent Benin 97/1 ASFV genotype I isolate at day 130 postimmunization. The levels of regulatory T cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.

Novel bacteriocins produced by Lactobacillus fermentum strains with bacteriostatic effects in milk against selected indicator microorganisms.

The aim of this work was to isolate and characterize bacteriocins produced by 2 Lactobacillus fermentum strains isolated from artisanal Mexican Cocido cheese. Fractions (F ≤3 kDa) obtained from cell-free supernatants of Lb. fermentum strains J23 and J32 were further fractionated by reversed-phase HPLC on a C18 column. Antimicrobial activities of F ≤3 kDa and bacteriocin-containing fractions (BCF), obtained from fractionation of F ≤3 kDa against 4 indicator microorganisms, were determined by the disk diffusion method and growth inhibition in milk. Subsequently, isolated BCF were analyzed by reversed-phase HPLC tandem mass spectrometry. Results showed that BCF presented antimicrobial activity against the 4 indicator microorganisms tested. For J23, one of the fractions (F3) presented the highest activity against Escherichia coli and was also inhibitory against Staphylococcus aureus, Listeria innocua, Salmonella Typhimurium, and Salmonella Choleraesuis. Similarly, fractions F3 and F4 produced by J32 presented antimicrobial activity against all indicator microorganisms. Furthermore, generation time and growth rate showed that F3 from J23 presented significantly higher antimicrobial activity against the 4 indicator microorganisms (2 gram-positive and 2 gram-negative) when inoculated in milk compared with F3 from J32. Interestingly, this fraction presented a broader antimicrobial spectrum in milk than nisin (control). Reversed-phase HPLC tandem mass spectrometry analysis revealed the presence of several peptides in BCF; however, F3 from J23 that was the most active fraction of all presented only 1 bacteriocin. The chemical characterization of this bacteriocin suggested that it was a novel peptide with 10 hydrophobic AA residues in its sequence and a molecular weight of 2,056 Da. This bacteriocin and its producing strain, J23, may find application as a biopreservative against these indicator microorganisms in dairy products.

Safety and Tolerability of More than Six Days of Tedizolid Treatment.

Tedizolid has demonstrated its efficacy and safety in clinical trials; however, data concerning its tolerability in long-term treatments are scarce. The aim of the study was to assess the indications and to describe the long-term safety profile of tedizolid. A multicentric retrospective study of patients who received tedizolid for more than 6 days was conducted. Adverse events (AEs) were identified from patients' medical records and laboratory data. The World Health Organization causality categories were used to discern AEs that were probably associated with tedizolid. Eighty-one patients, treated with tedizolid 200 mg once daily for a median (interquartile range [IQR]) duration of 28 (14 to 59) days, were included; 36 (44.4%) had previously received linezolid. The most common reasons for selecting tedizolid were to avoid linezolid potential toxicities or interactions (53.1%) or due to previous linezolid-related toxicities (27.2%). The most common indications were off-label, including prosthetic joint infections, osteomyelitis, and respiratory infections (77.8%). Overall, 9/81 patients (11.1%) experienced a probably associated AE. Two patients (2.5%) developed gastrointestinal disorders, 1 (1.2%) developed anemia, and 6 developed thrombocytopenia (7.4%) after a median (IQR) duration of treatment of 26.5 (17 to 58.5) days. Four (5%) patients discontinued tedizolid due to AEs. Among 23 patients with chronic renal failure (CRF), the rate of myelotoxicity was 17.4%, and only 8.7% had to stop tedizolid; 20 out of 22 with previous linezolid-associated toxicity had no AE. Long-term tedizolid treatments had good tolerance with rates of gastrointestinal AE and hematological toxicity lower than those reported with linezolid, particularly in patients with CRF and in those with a history of linezolid-associated toxicity.

PPARγ is critical for Mycobacterium tuberculosis induction of Mcl-1 and limitation of human macrophage apoptosis.

Peroxisome proliferator-activated receptor (PPAR)γ is a global transcriptional regulator associated with anti-inflammatory actions. It is highly expressed in alveolar macrophages (AMs), which are unable to clear the intracellular pathogen Mycobacterium tuberculosis (M.tb). Although M.tb infection induces PPARγ in human macrophages, which contributes to M.tb growth, the mechanisms underlying this are largely unknown. We undertook NanoString gene expression analysis to identify novel PPARγ effectors that condition macrophages to be more susceptible to M.tb infection. This revealed several genes that are differentially regulated in response to PPARγ silencing during M.tb infection, including the Bcl-2 family members Bax (pro-apoptotic) and Mcl-1 (pro-survival). Apoptosis is an important defense mechanism that prevents the growth of intracellular microbes, including M.tb, but is limited by virulent M.tb. This suggested that M.tb differentially regulates Mcl-1 and Bax expression through PPARγ to limit apoptosis. In support of this, gene and protein expression analysis revealed that Mcl-1 expression is driven by PPARγ during M.tb infection in human macrophages. Further, 15-lipoxygenase (15-LOX) is critical for PPARγ activity and Mcl-1 expression. We also determined that PPARγ and 15-LOX regulate macrophage apoptosis during M.tb infection, and that pre-clinical therapeutics that inhibit Mcl-1 activity significantly limit M.tb intracellular growth in both human macrophages and an in vitro TB granuloma model. In conclusion, identification of the novel PPARγ effector Mcl-1 has determined PPARγ and 15-LOX are critical regulators of apoptosis during M.tb infection and new potential targets for host-directed therapy for M.tb.

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

Flow Cytometry Data Preparation Guidelines for Improved Automated Phenotypic Analysis.

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103+ dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis.

Volatile and sensory evaluation of Mexican Fresco cheese as affected by specific wild Lactococcus lactis strains.

Lactococcus lactis is the lactic acid bacteria most frequently used for the production of cheese starter cultures, mainly because of their efficient production of aroma compounds. However, commercial cultures do not always produce the typical aroma notes of artisanal raw-milk cheeses. Thus, the objective of this study was to characterize the volatile compounds generated by wild L. lactis strains in Mexican Fresco cheese made with pasteurized milk. Four strains of wild L. lactis were evaluated for their aroma production in Mexican Fresco cheese using sensory and instrumental analysis. The aroma profiles were evaluated by descriptive sensory analysis. Volatiles were determined by solid-phase microextraction and gas chromatography-mass spectrometry. Principal component analysis was applied to interpret analytical and sensory data. Mexican Fresco cheese aroma was described as milkfat, yogurt, yeasty, barny, dirty socks, and Fresco cheese. Cheese with L. lactis strains R7 or B7 were most similar to commercial raw milk Fresco cheese in all aroma descriptors. Volatiles identified in all cheeses were esters, acids, alcohols, ketones, and aldehydes, but the main differences were found for total volatile relative abundance. Also, volatile concentrations (µg/g) in commercial raw milk Fresco cheese and cheeses made with L. lactis R7 or B7 were 4 methyl esters [C4 (4.15 vs. 5.47-13.74), C6 (0.12 vs. 1.53-15.34), C8 (1.06 vs. 0.32-6.65), and C10 (0.62 vs. 0.41-3.74)], 7 acids [C4 (1.92 vs. 0.30-9.29), C6-C10 (0.05-4.48 vs. 0.11-30.45), and C12 (0.13 vs. 0.28-0.30)], 2 alcohols [(3-methyl-1-butanol (3.48 vs. 3.4-13.13) and phenylethyl alcohol (0.10 vs. 0.63-2.04)], and 1 ketone (acetoin; 1.22 vs. 0.28-0.99). The first 3 principal components explained 78.2% of the total variation and clearly distinguished 3 main groups. Cheese made with L. lactis R7 was classified in the same group as key compounds associated with Fresco cheese aroma and show potential as a starter in Mexican Fresco cheese manufacture.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

Proof of Concept on Functionality Improvement of Mesenchymal Stem-Cells, in Postmenopausal Osteoporotic Women Treated with Teriparatide (PTH1-34), After Suffering Atypical Fractures.

Osteoporosis long-term treatment with nitrogen-containing bisphosphonates, has been associated with uncommon adverse effects, as atypical femoral fractures (AFF). Thus, treatment with teriparatide (TPTD; fragment of human parathyroid hormone; PTH1-34) has been proposed for such patients. Besides its anabolizing effect on bone, TPTD may affect stem-cell mobilization and expansion. Bone marrow mononuclear cells (BMMNC) were isolated from five women that had suffered AFF associated to bisphosphonate treatment, before and after 6 months of TPTD therapy. The presence of mesenchymal stromal cells (CD73, CD90 and CD105 positive cells), gene expression of NANOG, SOX2 and OCT4, proliferation, senescence and capacity to differentiate into osteoblasts and adipocytes were analyzed. After TPTD treatment, BMMNC positive cells for CD73, CD90 and CD105 increased from 6.5 to 37.5% (p < 0.05); NANOG, SOX2 and OCT4 were upregulated, being statistically significant for NANOG (p < 0.05), and cells increased proliferative capacity more than 50% at day 7 (p < 0.05). Senescence was reduced 2.5-fold (p < 0.05), increasing differentiation capacity into osteoblasts and adipocytes, with more than twice mineralization capacity of extracellular matrix or fat-droplet formation (p < 0.05), respectively. Results show that TPTD treatment caused BMMNC "rejuvenation", increasing the number of cells in a more undifferentiated stage, with higher differentiation potency. This effect may favor TPTD anabolic action on bone in such patients with AFF, increasing osteoblast precursor cells. Such response could also arise in other osteoporotic patients treated with TPTD, without previous AFF. Furthermore, our data suggest that TPTD effect on stromal cells may have clinical implications for bone-regenerative medicine. Further studies may deepen on this potential.