Gamma-delta T cells suppress microbial metabolites that activate striatal neurons and induce repetitive/compulsive behavior in mice.

Intestinal γδ T cells play an important role in shaping the gut microbiota, which is critical not only for maintaining intestinal homeostasis but also for controlling brain function and behavior. Here, we found that mice deficient for γδ T cells (γδ-/-) developed an abnormal pattern of repetitive/compulsive (R/C) behavior, which was dependent on the gut microbiota. Colonization of WT mice with γδ-/- microbiota induced R/C behavior whereas colonization of γδ-/- mice with WT microbiota abolished the R/C behavior. Moreover, γδ-/- mice had elevated levels of the microbial metabolite 3-phenylpropanoic acid in their cecum, which is a precursor to hippurate (HIP), a metabolite we found to be elevated in the CSF. HIP reaches the striatum and activates dopamine type 1 (D1R)-expressing neurons, leading to R/C behavior. Altogether, these data suggest that intestinal γδ T cells shape the gut microbiota and their metabolites and prevent dysfunctions of the striatum associated with behavior modulation.

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Gamma-delta T cells modulate the microbiota and fecal micro-RNAs to maintain mucosal tolerance.

BACKGROUND: Gamma-delta (γδ) T cells are a major cell population in the intestinal mucosa and are key mediators of mucosal tolerance and microbiota composition. Little is known about the mechanisms by which intestinal γδ T cells interact with the gut microbiota to maintain tolerance. RESULTS: We found that antibiotic treatment impaired oral tolerance and depleted intestinal γδ T cells, suggesting that the gut microbiota is necessary to maintain γδ T cells. We also found that mice deficient for γδ T cells (γδ-/-) had an altered microbiota composition that led to small intestine (SI) immune dysregulation and impaired tolerance. Accordingly, colonizing WT mice with γδ-/- microbiota resulted in SI immune dysregulation and loss of tolerance whereas colonizing γδ-/- mice with WT microbiota normalized mucosal immune responses and restored mucosal tolerance. Moreover, we found that SI γδ T cells shaped the gut microbiota and regulated intestinal homeostasis by secreting the fecal micro-RNA let-7f. Importantly, oral administration of let-7f to γδ-/- mice rescued mucosal tolerance by promoting the growth of the γδ-/--microbiota-depleted microbe Ruminococcus gnavus. CONCLUSIONS: Taken together, we demonstrate that γδ T cell-selected microbiota is necessary and sufficient to promote mucosal tolerance, is mediated in part by γδ T cell secretion of fecal micro-RNAs, and is mechanistically linked to restoration of mucosal immune responses. Video Abstract.

Colorectal cancer: comparative analysis between two series of patients separated by more than three decades

Abstract Aim This study characterizes Colorectal Cancer (CRC) incidence in the University Hospital Ramon and Cajal, Madrid, and analyzes variations over time. It establishes risk groups, aiming to discover whether diagnosis can be determined in less advanced stages of disease. Method Evolutionary epidemiological study of genetic and environmental factors contributing to the development of CRC in this district that enables the comparison of two cohorts of patients separated by 37 years: G1 (patients of current group) and G2 (patients of historical group). The main risk variables gleaned retrospectively were analyzed and the statistical association between cohorts was determined. Results The mean age of patients increased significantly from 64 to 71 along with the incidence of ascending colon cancer. G1 scored higher than G2 for: the incidence of colon cancer in men, detection of adenomatous polyps (48.1%), percentage of resectability with curative intent (80.4%), and Dukes A stage (34.1%) (p < 0.001). Conclusion Biological aspects of CRC have been compared against its profile three decades earlier. We can confirm the existence of concrete changes in the manifestation and staging at the time of diagnosis or following earlier treatment. (AU)

PD-L1+ and XCR1+ dendritic cells are region-specific regulators of gut homeostasis.

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.

Colocación intraoperatoria anterógrada de stent pancreático y plastia de ligamento redondo para prevenir la fístula pancreática tras la pancreatectomía distal / Anterograde intraoperative pancreatic stent placement and round ligament patch to prevent pancreatic fistula after distal pancreatectomy

La fístula pancreática tras la pancreatectomía distal supone una de las complicaciones más frecuentes y con mayor morbilidad asociada en este tipo de cirugía. Hasta el momento ningún método empleado se ha mostrado eficaz en reducir la fístula pancreática de forma contrastada. Presentamos el desarrollo de una nueva técnica quirúrgica que combina la colocación del stent pancreático junto con la colocación de parche autólogo de ligamento redondo sobre el borde pancreático. Tras la realización de la transección pancreática, se introduce de forma anterógrada la prótesis pancreática previo paso de una guía. Se comprueba la colocación de la prótesis y se cierra el conducto de Wirsung. Por último, se realiza una plastia autóloga de ligamento falciforme. A las 6-8 semanas se retira la prótesis vía endoscópica. La técnica ha sido empleada en 2 pacientes sin presentar fístula pancreática postoperatoria. Esta técnica combinada pretende introducir un nuevo elemento en la prevención de la fístula pancreática mediante una técnica reproducible sin una dificultad técnica añadida. (AU)

Postoperative pancreatic fistula in distal pancreatectomy is one of the most important complications in this surgery and it is associated with high morbidity and mortality. Pancreatic fistula after distal pancreatectomy remains an unsolved problem and none preventive procedure has been shown effectively. We present a new technique that combine pancreatic stent placement with round ligament autologous patch over pancreatic edge. A guide is introduced through Wirsung duct prior to stent placement. After stent assessment, Wirsung duct is closed. Finally, falciform ligament autologous patch is placed over pancreatic edge. After 6-8 weeks, the stent is removed by oral endoscopy. This technique introduces a new issue on the pancreatic fistula prevention. (AU)

Anterograde intraoperative pancreatic stent placement and round ligament patch to prevent pancreatic fistula after distal pancreatectomy. / Colocación intraoperatoria anterógrada de stent pancreático y plastia de ligamento redondo para prevenir la fístula pancreática tras la pancreatectomía distal.

Postoperative pancreatic fistula in distal pancreatectomy is one of the most important complications in this surgery and it is associated with high morbidity and mortality. Pancreatic fistula after distal pancreatectomy remains an unsolved problem and none preventive procedure has been shown effectively. We present a new technique that combine pancreatic stent placement with round ligament autologous patch over pancreatic edge. A guide is introduced through Wirsung duct prior to stent placement. After stent assessment, Wirsung duct is closed. Finally, falciform ligament autologous patch is placed over pancreatic edge. After 6-8 weeks, the stent is removed by oral endoscopy. This technique introduces a new issue on the pancreatic fistula prevention.

Assessment of potential reference sites for evaluating the ecological status of subtropical and temperate Brazilian lotic systems using the epilithic diatom community.

This research aimed at determining reference sites in southern Brazilian lotic systems, using the Water Quality Trophic Index (WQTI), based on epilithic diatom communities. Within the period of 2012 and 2016, 140 epilithic diatom samples from seven sampling sites within the spring area of the Andreas Stream Hydrographic Basin, Vera Cruz County, RS, Brazil, were analyzed quarterly. Similarly, from 2012 and 2014, water samples in these sampling sites were collected for determination of physicochemical and microbiological parameters, such as water temperature, pH, turbidity, dissolved oxygen, biochemical oxygen demand, nitrate, phosphate, total dissolved solids, and fecal coliforms. The results indicated the occurrence of two diatom samples groups with significant differences (p < 0.05). Group 1 (sampling sites P1, P4, and P5) was classified in the oligotrophic category with an average WQTI of 1.3 ± 0.2, while group 2 (sampling sites P2, P3, P6, and P7) was classified in the ß-mesotrophic category with an average value of 2.0 ± 0.4). Achnanthidium minutissimum and Platessa hustedtii the dominant species were classified as oligotrophic taxa. Thus, we proposed the upper limit of the first interval of the WQTI score scale, equal to 1.5, as a reference value to differentiate the limits between the "high" and "good" ecological status to determine "reference sites" for subtropical and temperate Brazilian aquatic systems. The physical, chemical, and microbiological water quality evaluation gives support to this proposal, as far as the results indicated a significant improvement in the water quality, classifying sampling sites P1, P4, and P5 as having good quality appropriate for multiple uses.

Development of the Diatom Ecological Quality Index (DEQI) for peri-urban mountain streams in the Basin of Mexico.

In the Basin of Mexico, one of the most important economic regions in the country with over 22 million inhabitants, peri-urban streams have been transformed into sewers, drains, and pipes to avoid flooding or unsanitary conditions; the change affects not only the ecosystem's goods and services but also the aquatic communities that support the natural ecological processes. We aimed to develop a diatom-based diagnosis of the ecological quality of these aquatic ecosystems through the study of epilithic diatom response to regional environmental gradients. Samples of epilithic diatoms and water were collected in 45 sites representing 12 perennial streams, and multivariate analyses were performed on environmental and biological data. The ecological quality gradient to which diatoms responded was related to oxygen saturation, soluble reactive phosphorous, dissolved inorganic nitrogen, and hydromorphological quality. Three species groups were recognized according to their ecological preferences along CCA1 axis, indicators of high and low ecological quality, and tolerant species. By assigning an indicator value to each species group, we calculated the DEQI using the formula adapted from Pantle and Buck, indicating five different ecological quality classes. This index is proposed for complementing the ecological quality evaluation as a biological metric that responds to multiple regional stressors of the structure and function of these peri-urban streams in the Basin of Mexico.

Potential for bifenthrin removal using microalgae from a natural source.

The accumulation of environmental pesticides can cause problems in aquatic ecosystems and adverse effects in humans. These compounds can be found in water due to runoff from agricultural, industrial and domestic applications. In Southern Brazil, tobacco cultivation is one of the most important economic agricultural activities. The bifenthrin pesticide, classified as having moderate toxicity (class II), is commonly used as an insecticide in this culture. In this context, the present research aimed to study the performance of microalgae-induced bioremediation processes. Microalgae were isolated from a natural water source in the city of Santa Cruz do Sul, RS, Brazil, which is an artificial reservoir used for public water supply. For this purpose, biodegradation, biosorption, influence of pH, percentage of inoculum and photoperiod were evaluated in batch experiments for 20 cultivation days. After the phycoremediation process, the bifenthrin pesticide (m/z = 181) was quantified by gas chromatography with mass spectrometry (GC-MS). The results indicated that microalgae isolated from the water of the lake were able to contribute to the removal of approximately 99% of bifenthrin through biodegradation and biosorption processes. Photodegradation was identified (>77%) and the best condition for the phycoremediation was 20% inoculum with a photoperiod of 18:6 h.

Genetic barcoding of Ecuadorian epilithic diatom species suitable as water quality bioindicators.

Diatom identification is a key step in using these microorganisms as water quality bioindicators. Morphological diagnosis is a difficult task due to the enormous number of species and their microscopic size. This can be overcome using molecular tools to complement the diagnosis. The main goal of this work was to obtain the DNA barcode of Ecuadorian epilithic diatoms with a wide geographical distribution, a well-defined ecological range and characteristics that allow them to be reliable indicator species. Unialgal diatom cultures were obtained from environmental samples of Ecuadorian Andean streams. Morphological characterization of cultures was carried out under SEM microscopy. For molecular characterization, 18SV4 and rbcL barcodes were sequenced from each strain and blasted against a GenBank database. A phylogenetic tree for each barcode was constructed using the ML method including sequences of strains of the studied species from different geographical locations. The results showed the following five species to be suitable as bioindicators and these were isolated. Sellaphora seminulum (strain JA01b, c), Nitzschia fonticola (strain SP02a) and N. palea (strain CA01a) are tolerant to eutrophication; Eolimna minima (strain CH02a) is a mesotrophic water bioindicator, and Achnanthidium minutissimum (strain JA01a) is an oligotrophic water bioindicator. The comparison with the GenBank database of the barcoding regions supported the morphological identification. The barcoding sequences of the strains showed a high percentage of identity with the sequences reported in INSDC databases for the same species. The topology of the phylogenetic trees demonstrates that epilithic diatoms from Ecuador are closely related to those of same species isolated from other geographical regions. This study is a first attempt to establish a morphological and molecular taxonomic reference library for neotropical diatoms. This study demonstrates that it would be feasible to use the existing barcoding data for diatoms to develop molecular tools for the bioassessment of aquatic ecosystems in the Ecuadorian Andean region.

L'identification des diatomées est une étape clé dans l'utilisation de ces microorganismes comme bioindicateurs de la qualité de l'eau. Le diagnostic morphologique est une tâche difficile en raison du nombre considérable d'espèces et de leur dimension microscopique. Il est possible de surmonter cette difficulté en utilisant des techniques moléculaires pour compléter le diagnostic. L'objectif principal de ce travail était d'obtenir le code-barre de l'ADN des diatomées épilithiques équatoriennes ayant une large distribution géographique, une niche écologique bien définie et des caractéristiques leur permettant d'être des espèces indicatrices fiables. Des cultures de diatomées unialgales ont été obtenues à partir d'échantillons environnementaux de cours d'eau des Andes équatoriennes. La caractérisation morphologique des cultures a été réalisée sous microscopie MEB. Pour la caractérisation moléculaire, les codes-barres 18SV4 et rbcL ont été séquencés à partir de chaque souche et comparés à la base de données GenBank. Pour chaque code-barres, un arbre phylogénétique a été construit à partir de la méthode ML comprenant des séquences de souches des espèces étudiées, provenant de différents lieux géographiques. Les résultats ayant montré que les cinq espèces suivantes étaient appropriées comme bioindicateurs, elles ont été isolées. Sellaphora seminulum (souche JA01b, c), Nitzschia fonticola (souche SP02a) et N. palea (souche CA01a) sont tolérantes à l'eutrophisation ; Eolimna minima (souche CH02a) est un bioindicateur d'eau mésotrophe, et Achnanthidium minutissimum (souche JA01a) est un bioindicateur d'eau oligotrophe. La comparaison avec la base de données GenBank des régions de code-barres a supporté leurs identifications morphologiques. Les séquences de code-barres des souches ont montré un pourcentage élevé d'identité génétique avec les séquences signalées dans les bases de données de l'INSDC pour la même espèce. La topologie des arbres phylogénétiques démontre que les diatomées épilithiques de l'Équateur sont étroitement liées à celles des mêmes espèces isolées d'autres régions géographiques. Cette étude est une première tentative d'établir une bibliothèque de référence morphologique et taxonomique moléculaire pour les diatomées néotropicales. Cette étude démontre qu'il serait possible d'utiliser les données de code-barres existantes pour les diatomées afin de développer des instruments moléculaires pour la bioévaluation des écosystèmes aquatiques dans la région andine équatorienne.

Toxicity evaluation of hospital laundry wastewaters treated by microbial fuel cells and constructed wetlands.

Hospital laundries generate high wastewater volumes with the presence of several contaminants. Nevertheless, few studies have investigated the toxicity of these effluents and looked for treatment alternatives that might reduce this eventual toxicity. So, the present study assessed the performance of an integrated system combining a microbial fuel cell (MBFC) and a constructed wetland (CW) to reduce toxic effects of wastewaters generated at a hospital laundry. After collection, raw effluents remained 7 days at the first unit (MBFC) of integrated system. Afterward, they were transferred to the second unit (CW) unit where remained more 7 days totaling a hydraulic detention time of 14 days. The toxicity evaluation involved three different organisms: Daphnia magna (acute ecotoxicity), Lactuca sativa (phytotoxicity) and Allium cepa (phytotoxicity, cytotoxicity, mutagenicity, and genotoxicity). Got results revealed an extremely acute ecotoxicity against D. magna, high phytotoxic effects in the L. sativa and A. cepa assays, and genotoxicity in the A. cepa assay for the untreated effluents. Furthermore, no significant incidence of micronuclei was observed in the raw wastewaters. Regarding the treatment, after the first stage, it was possible to verify that MBFC reduced the toxicity of the wastewaters only in some tested assays (endpoints) while after the CW (second stage) the effluents presented a complete absence of toxicity of the investigated bioassays. Therefore, the use of the integrated system combining two environmentally friendly technologies can be considered promising, since both MBFC and CW presented a complimentary effect with excellent results regarding the reduction of the overall toxicity of hospital laundry wastewaters.

Relevancia del margen de resección positivo en adenocarcinoma ductal de páncreas y otros factores pronósticos / Relevance of positive resection margins in ductal pancreatic adenocarcinoma and prognostic factors

Introducción: Actualmente en cirugía del cáncer de páncreas se considera margen de resección afecto (R1) la presencia de células tumorales a <1mm del borde de resección. El objetivo principal del estudio es analizar el impacto del margen de resección en la supervivencia. Métodos: Análisis retrospectivo con análisis de regresión multivariante de una base de datos prospectiva (2008-2017), donde se incluye el margen de resección, el margen de resección ampliado (R1 < 1 mm), la resección vascular, la afectación linfática, las complicaciones quirúrgicas, la diferenciación tumoral y el tratamiento adyuvante. Resultados: Un total de 80 pacientes fueron analizados, 42 (52%) R1 y 38 (48%) R0. No se encontraron diferencias en la composición de ambos grupos salvo en la resección vascular, que fue mayor en el grupo R1, 12 (21%) vs. 2 (3%). La supervivencia en el grupo R0 fue de 19 meses vs. 24 meses en el grupo R1 (p = 0,13). El margen ampliado (R1 < 1 mm) tuvo una supervivencia de 21 meses vs. 31 meses en R0 ampliado (p = 0,55). En el análisis multivariante solo se encontraron la afectación ganglionar (p = 0,02; HR = 2,88), la diferenciación tumoral (p = 0,02; HR = 3,2) y la adyuvancia (p < 0,01; HR = 0,21) como factores pronósticos de supervivencia. Conclusiones: En el estudio la resección R1 no supone un factor pronóstico. La afectación ganglionar, el grado de diferenciación y el tratamiento adyuvante son factores pronósticos. Debe demostrarse el beneficio de ampliar los márgenes de resección. Son necesarios más estudios para valorar el impacto del margen de resección

Introduction: Currently, R1 resection is defined by the presence of tumor cells within < 1 mm of the resection margin. The main aim of this study was to analyze the impact of positive margins (R1) on survival outcomes in pancreatic cancer. Methods: We performed a retrospective analysis with multivariate regression analysis of a prospective database from 2008-2017, which included resection margin status, expanded resection margin (R1 < 1 mm), vascular resection, lymphatic involvement, surgical complications, tumor differentiation grade and adjuvant treatment. Results: A total of 80 patients were analyzed: 42 (52%) R1; 38 (48%) R0. No differences were found in the composition of the two groups except for the vascular resection, which was more frequent in the R1 group: 12 (21%) vs 2 (3%). Overall survival in the R0 group was 19 months vs 24 months in the R1 group (p = 0.13). Wide R1 (R1 < 1 mm) had an overall survival of 21 months versus 31 months in wide R0 (p = 0.55). In the multivariate analysis, only lymph node involvement (p = 0.02, HR = 2.88), tumor differentiation (p = 0.02, HR = 3.2) and adjuvant therapy (p < 0.01; HR = 0.21) were found to be factors related to survival. Conclusions: R1 resection is not an independent risk factor. Lymph node involvement, differentiation grade and adjuvant treatment are prognostic factors. The benefit of expanding the resection margins should be demonstrated. More studies are needed to assess the impact of the resection margin

Biomonitoring of urban wastewaters treated by an integrated system combining microalgae and constructed wetlands.

The objectives of the present study were to apply different, toxicological assays for monitoring the toxicity of treated and untreated urban effluents produced at a university campus. The research was conducted at the wastewater treatment plant of the University of Santa Cruz do Sul, (UNISC), from october 2018 to april 2019. An integrated system with, anaerobic reactor (AR), microalgae (MA) and constructed wetlands (CWs) was, proposed for detoxification of the wastewaters produced at the university campus with a hydraulic detention time of 17 days. Daphnia, magna (ecotoxicity) and Allium cepa (phytotoxicity, cytotoxicity, and, genotoxicity) were used as tools to monitor the efficiency of the integrated system. Obtained results showed that the integrated system (MA, + CWs) presented good COD and BOD5 reductions, besides removal rates of, almost 98% for N-NH3, being much more efficient than the UNISC wastewater, treatment plant (UWTTP). The results of ecotoxicity presented the raw wastewaters (RW) as slightly toxic and an absence of ecotoxicity in all the treatments steps. Regarding phytotoxicity, the results showed no significant differences between the treatments. The cytogenetic assays indicated a significant increase in mitotic index (MI) (p < 0.001) after treatment by CWs compared to the final treatment UWTTP while the results, regarding binucleated cells (BNC) did not present significant differences, among the treatments. Micronucleus (MN) indexes were significantly different between the UWWPT and the integrated system (p < 0.01). In relation to chromosome aberrations (CA) the results indicate a significant difference between the CWs and UWWTP treatments (p < 0.01) and, RW and CWs (p < 0.001), confirming the detoxifying potential of the integrated system when compared to UWWPT. Thus, the results of the present research highlight the relevance in the proposition of the integrated system as an alternative of cleaner technology to the detriment of conventional technologies applied in wastewater treatment.