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N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

Abstract The plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses.

Abstract Human intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a framework combining single‐cell RNA‐Seq and highly multiplex RNA FISH and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages and induces expression of the cell proliferation marker MKI67. Intriguingly, each intestinal epithelial cell lineage exhibits a unique basal expression of interferon‐stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon‐stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our framework is applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

Abstract We aimed at identifying the developmental stage at which leukemic cells of pediatric T‐ALLs are arrested and at defining leukemogenic mechanisms based on ATAC‐Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T‐cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T‐cell development. Deconvolution using signature regions revealed that T‐ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF‐binding motif profiles. We integrated ATAC‐Seq and RNA‐Seq and found DAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper‐accessible in T‐ALLs. DAB1‐negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper‐accessible binding sites for SPI1 (PU.1), a TF crucial for normal T‐cell maturation. In conclusion, our analyses of chromatin accessibility and TF‐binding motifs showed that pediatric T‐ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest.

Listeria monocytogenes is a pathogen difficult to control, due to its resistance to extreme conditions. The antimicrobial activity of a mixture of metabolites produced by lactic acid bacteria was evaluated against L. monocytogenes. Bacterial combined cultures in 1:1 ratio of Lactobacillus plantarum and Weissella cibaria (treatment LP + WC) and mixtures in ratio 1:1:1 of Lactobacillus brevis, L. plantarum, and W. cibaria, (treatment (LB + LP + WC) were grown by discontinuous fermentation, at 32 °C for 48 h. At 1, 2, 6, 12, 24 and 48 h of fermentation, samples were taken, the biomass was separated from the metabolites, and the antimicrobial activity of the metabolites was measured in vitro against L. monocytogenes. For comparison, experimental data published in the literature corresponding to monocultures of L. brevis (L.B), L. plantarum (LP) and W. cibaria (WC) were used. The antimicrobial activity was measured by a surface diffusion technique using absorbent paper discs impregnated with 60 μl from each metabolite and placed on the TSA agar surface (36 °C, 24 h). The metabolites from the microbial mixtures showed statistical differences with respect to their respective monocultures. With the treatment (LP + WC) an inhibition diameter of 2.54 cm was obtained at 12 h of fermentation, this value was higher than those obtained in the monoculture LP (2.19 cm), and WC (2.44 cm), during the same period. In the mixture (LB + LP + WC) during the first 12 h of fermentation, the antimicrobial activity was higher (2.12–2.28 cm) than the antimicrobial activity of the monoculture LB (1.66–2.23 cm). The use of metabolites from the co-culture of L brevis, L. plantarum and W. cibaria under the evaluated conditions, potentiate the antimicrobial activity of L. brevis against L. monocytogenes, therefore, they are promising in bio-preservation.

En Colombia, se han implementado Sistemas de Transporte Masivo en varias ciudades del País en los últimos 4 años; se desconoce la magnitud y comportamiento de las ausencias laborales en esta actividad económica y grupo de trabajadores. Objetivo: Describir el comportamiento del ausentismo laboral por motivos de salud en Operadores de una empresa de buses del Sistema de Transporte Masivo en la ciudad de Cali, durante el periodo 2010 al 2013. Metodología: descriptiva longitudinal retrospectiva, con base en 2700 incapacidades generadas por motivos de salud. Resultados: Se evidenció que con la edad se incrementa la proporción de personas ausentes, excepto para los grupos de edad de 44 – 49 años. Durante el período de estudio del total de tiempo programado el total de tiempo perdido fue el 2.3%. En cuanto al tipo de contingencia La “Enfermedad General” concentró el 97,66% de los eventos y fue responsable de la más alta frecuencia de días perdidos y de todos los indicadores. Conclusiones: Los resultados de este estudio demuestran la necesidad de la investigación de las causas raíz por parte de la empresa, de los tres principales grupos diagnósticos: Enfermedades infecciosas y parasitarias, enfermedades del sistema respiratorio y enfermedades del sistema osteomuscular que generaron el mayor número de ausencias en los trabajadores y que ocasionan el mayor índice de enfermedad general.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.