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Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfK

Supplementary Table 2 from Identification of Secreted Proteins that Mediate Cell-Cell Interactions in an In vitro Model of the Lung Cancer Microenvironment

Supplementary Table 1 from Identification of Secreted Proteins that Mediate Cell-Cell Interactions in an In vitro Model of the Lung Cancer Microenvironment

Supplementary Figures 1-5 from Identification of Secreted Proteins that Mediate Cell-Cell Interactions in an In vitro Model of the Lung Cancer Microenvironment

Funding: This work was supported by the Leverhulme Trust (Research project grant 2015-268 to NA, RK, and KP) and the UK Natural Environment Research Council (NE/S001417/1) to NA, KP, RK, MC and AF. The Raman Microscope is supported by the EPSRC (Light Element Analysis Facility Grant EP/T019298/1 and Strategic Equipment Resource Grant EP/R023751/1). Resolving how factors such as temperature, pH, biomolecules and mineral growth rate influence the geochemistry and structure of biogenic CaCO3, is essential to the effective development of palaeoproxies. Here we optimise a method to precipitate the CaCO3 polymorph aragonite from seawater, under tightly controlled conditions that simulate the saturation state (Ω) of coral calcification fluids. We then use the method to explore the influence of aspartic acid (one of the most abundant amino acids in coral skeletons) on aragonite structure and morphology. Using ≥200 mg of aragonite seed (surface area 0.84 m2), to provide a surface for mineral growth, in a 330 mL seawater volume, generates reproducible estimates of precipitation rate over Ωaragonite = 6.9-19.2. However, unseeded precipitations are highly variable in duration and do not provide consistent estimates of precipitation rate. Low concentrations of aspartic acid (1-10 µM) promote aragonite formation, but high concentrations (≥ 1 mM) inhibit precipitation. The Raman spectra of aragonite precipitated in vitro can be separated from the signature of the starting seed by ensuring that at least 60% of the analysed aragonite is precipitated in vitro (equivalent to using a seed of 200 mg and precipitating 300 mg aragonite in vitro). Aspartic acid concentrations ≥ 1mM caused a significant increase in the full width half maxima of the Raman aragonite v1 peak, reflective of increased rotational disorder in the aragonite structure. Changes in the organic content of coral skeletons can drive variations in the FWHM of the Raman aragonite ν1 peak, and if not accounted for, may confuse the interpretation of calcification fluid saturation state from this parameter. Publisher PDF

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σ(ECF)) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σ(ECF) factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σ(ECF) factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σ(ECF)). Here, we describe a new σ(ECF) factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σ(ECF) factors in phytopathogenic bacteria.

Autochthonous yeasts of oenological origin are adapted to highly stressful and selective environments, which makes them potential candidates for probiotics. The objective of the present study was to explore the probiotic potential of 96 native yeasts of oenological origin, their biosafety, resistance to gastrointestinal tract conditions and adhesion properties. Regarding biosafety, 66 isolates shown negative hemolytic activity, negative urease activity and susceptibility to 3 or more antifungals. After the gastrointestinal resistance test, 15 isolates were selected that showed growth at different temperatures, tolerance to low pH and the presence of bile salts in in vitro tests. In general, survival after simulated conditions of the gastrointestinal tract was high and more restrictive was the duodenal. The results of the adhesion properties showed highly variable hydrophobicity and a high percentage of autoaggregation at 24 h. The maximum production of biofilm was detected in the Pichia strains. Of a total of 96 yeast strains, 15 non-Saccharomyces yeasts presented suitable properties as probiotic candidates. The native winemaking strains performed better than the reference probiotic strain, Saccharomyces cerevisiae var. boulardii CNCM I-745, which reaffirms that these strains are promising probiotic candidates and further studies are necessary to confirm their probiosis.

Bacteria of the Xanthomonas genus are mainly phytopathogens of a large variety of crops of economic importance worldwide. Xanthomonas spp. rely on an arsenal of protein effectors, toxins and adhesins to adapt to the environment, compete with other microorganisms and colonize plant hosts, often causing disease. These protein effectors are mainly delivered to their targets by the action of bacterial secretion systems, dedicated multiprotein complexes that translocate proteins to the extracellular environment or directly into eukaryotic and prokaryotic cells. Type I to type VI secretion systems have been identified in Xanthomonas genomes. Recent studies have unravelled the diverse roles played by the distinct types of secretion systems in adaptation and virulence in xanthomonads, unveiling new aspects of their biology. In addition, genome sequence information from a wide range of Xanthomonas species and pathovars have become available recently, uncovering a heterogeneous distribution of the distinct families of secretion systems within the genus. In this review, we describe the architecture and mode of action of bacterial type I to type VI secretion systems and the distribution and functions associated with these important nanoweapons within the Xanthomonas genus.

The Chacopampean plain is one of the most arsenic affected areas in Argentina, especially in groundwater, used both for animal drinking and forage irrigation. The main objective of this study was to determine the effect of arsenic (As) present in irrigation water on the germination parameters of red clover (Trifolium pratense L.), white clover (Trifolium repens L.), and alfalfa (Medicago sativa L.) seeds exposed to different concentrations of As(V). The germination and viability parameters of seeds from the three species were not affected by elevated concentrations of As. However, As significantly reduced the radicle and hypocotyl lengths of the three forage specimens. The inhibition level of the plants increased with the increase in the concentration of As. Regarding to hypocotyl length/radicle length ratio, the results evidenced that both the hypocotyl and radicle of clover species are affected in the same degree, while alfalfa radicles seemed more affected than hypocotyls at higher concentrations of As. Our results showed that irrigation with solutions containing As affect seedling growth parameters differently in the three species, and the effect of As is mostly evidenced when in high concentrations. A planície de Chacopampeana é uma das áreas mais afetadas pelo arsênico na Argentina, principalmente em águas subterrâneas, usada tanto para água potável animal quanto para irrigação forrageira. O principal objetivo deste estudo é determinar o efeito do arsênio (As) presente na água de irrigação sobre os parâmetros de germinação de sementes forrageiras: trevo vermelho (Trifolium pratense L.), trevo branco (Trifolium repens L.) e alfafa (Medicago sativa L.) expostos a diferentes concentrações de As (V). Verificouse que os parâmetros de germinação e viabilidade das sementes das três espécies não foram afetados pelas concentrações elevadas de As. No entanto, reduziu significativamente o comprimento da radícula e o comprimento do hipocótilo das três mudas de forragem, e seu nível de inibição é maior com o aumento da concentração de As. Quanto à relação comprimento de hipocótilo/comprimento da radícula, os resultados evidenciaram que nas espécies de trevo tanto o hipocótilo quanto a radícula são afetados no mesmo grau, enquanto as radículas de alfafa pareceram mais afetadas do que os hipocótilos em concentrações maiores de As. Nossos resultados mostraram que a irrigação com soluções contém Afetar os parâmetros de crescimento de plântulas de maneira diferente nas três espécies, e foi demonstrado que o efeito do As é principalmente evidenciado em altas concentrações. Fil: Alvarez Gonçalvez, Cristina Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Rodriguez, Magali. Universidad de Buenos Aires; Argentina Fil: Fernandez Cirelli, Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Pérez Carrera, Alejo Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina

The overlap between family and business systems creates a particular bundle of resources, which is a specific familiness that may determine how family firms positively or negatively manage their conflicts. In this article, we review the current research on conflict management and family firms and suggest theoretical propositions about the influence of familiness in constructive conflict management in family firms. We propose that specific levels of structural, cognitive, and relational dimensions of familiness configure a specific arrangement of resources that we conceptualized as collaborative familiness, which enhances constructive conflict. We discuss the main implications of this conceptualization in terms of its theoretical contributions, further research, and practice.

El plátano es uno de los productos más consumidos en el mundo, para el año 2019 se cosecharon 107.941.444 toneladas aproximadamente, producción en la cual Colombia aporta el 2,7%. A nivel nacional, el plátano es consumido en fresco y procesado industrialmente en la elaboración de snacks fritos, con contenidos de grasa de alrededor del 40% con base en su peso; productos catalogados dentro del grupo de alimentos no saludables. El uso combinado de tecnologías novedosas, como el tratamiento con biopeliculas y fritura por inmersión al vacío genera una alternativa para producción de snacks con menos contenido de aceite acompañado de parámetros de calidad aceptables y características organolépticas agradables para los consumidores El objetivo del trabajo fue evaluar la fritura por inmersión de rodajas de plátano verde (con ºBrix de 4,9±0,7) recubiertas con goma guar (1,4% p/v) en distintas condiciones de proceso bajo el análisis de superficie de respuesta utilizando como metodología el diseño central compuesto rotable, considerando la presión (30-62) kPa, tiempo (180-360) s y fuerza impulsora (43-73) ºC. El óptimo alcanzado para minimizar el contenido de aceite fue: presión de 73 kPa, tiempo de 341 s y fuerza impulsora 43°C. Los resultados obtenidos como óptimo estadístico en el análisis sensorial del snack recubierto señalaron una aceptabilidad del 55% del producto obtenido.

Simple Summary Dengue is an important and growing public health problem. To date, no specific therapeutic or effective prophylactic measures exist. Therefore, vector control remains the primary approach to prevent dengue virus (DENV) infection in humans. Recent findings highlight that viruses regulate mitochondrial function and dynamics to facilitate viral proliferation. In this study, we report that DENV infection modulates mitochondrial physiology in C6/36 mosquito cells. Our results revealed that DENV alters redox metabolism and mitochondrial membrane potential without any significant change in cellular ATP pool or viability. In addition, we observed preservation of the respiratory control ratio and translocation of mitofusins to mitochondria. These results suggest that mitochondrial fusion could be required for the maintenance of mitochondrial function in C6/36 mosquito cells infected with DENV. Abstract Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

This study investigated the relationship between phytoplankton biomass and taxonomic composition and hydrographic variables in the Northeastern Tropical Pacific for June 2015, March 2016, and September 2016. Hydrographic data were measured between surface and 100 m; samples were collected at the surface and the deep chlorophyll maximum (DCM) along one or two transects. Pigments were quantified by high-performance liquid chromatography; the CHEMTAX software was used to determine the relative contribution to chlorophyll a of the main taxonomic groups. Our results show that the studied region is characterized by a stable vertical distribution of phytoplankton biomass regardless of the season (winter, spring or summer). A subsurface maximum (DCM) is always observed close to the bottom of the mixed layer where there is a higher abundance of larger groups (diatoms and Prymnesiophytes) while at the surface community, composition was generally dominated by picoplankton (Cyanobacteria and Prochlorococcus), and occasionally Prymnesiophytes. However, during the spring cruise (June 2015), affected by the 2015–2016 El Nino, phytoplankton biomass at the DCM markedly decreased along with an increase in the abundance of Chlorophytes. On the other hand, in September 2016, there was an unexpected increase in phytoplankton biomass at the DCM, although stratification was strong, and Prymnesiophytes comprised 60% of the community at the surface. The evaluation of nutrient and light will be necessary in future studies to determine their role in this temporal variability. Finally, chemotaxonomy (HPLC/CHEMTAX) proved to be a valuable tool for describing phytoplankton distribution at group level in this region and its relationship with physical processes.

Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.

Family firms are a unique setting to study constructive conflict management due to the influence of family ties of the owning family imprinting a sense of common purpose and shared destiny, and high levels of trust. We study the relationship between shared vision and trust that intervene in the adoption of constructive conflict management. To achieve our purpose, we carried out a systematic indirect observation using a mixed methods approach. We used the narratives of 17 semi-structured interviews, audio-recorded and transcribed, of family and non-family managers or directors from five Spanish family firms in the siblings' partnership stage, combined with documentary data obtained from different sources. Intra- and inter-observer reliability were confirmed. Results show a dynamic relationship between shared vision and specific components of trust (benevolence and ability) at different levels of conflict management. We also provide evidence of specific processes of concurrence-seeking and open-mindedness in family and ownership forums accounting for the relevance of family governance in these type of organizations. Family firms are a sum of several subsystems which exhibit a particular resources configuration. This study sheds light on constructive conflict management in family firms opening interesting avenues for further research and offering practical implications to managers, owners, and advisors. ispartof: FRONTIERS IN PSYCHOLOGY vol:12 ispartof: location:Switzerland status: published

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60–93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.

Graphical abstract, Bacteria of the Xanthomonas genus are mainly phytopathogens of a large variety of crops of economic importance worldwide. Xanthomonas spp. rely on an arsenal of protein effectors, toxins and adhesins to adapt to the environment, compete with other microorganisms and colonize plant hosts, often causing disease. These protein effectors are mainly delivered to their targets by the action of bacterial secretion systems, dedicated multiprotein complexes that translocate proteins to the extracellular environment or directly into eukaryotic and prokaryotic cells. Type I to type VI secretion systems have been identified in Xanthomonas genomes. Recent studies have unravelled the diverse roles played by the distinct types of secretion systems in adaptation and virulence in xanthomonads, unveiling new aspects of their biology. In addition, genome sequence information from a wide range of Xanthomonas species and pathovars have become available recently, uncovering a heterogeneous distribution of the distinct families of secretion systems within the genus. In this review, we describe the architecture and mode of action of bacterial type I to type VI secretion systems and the distribution and functions associated with these important nanoweapons within the Xanthomonas genus.

Iron coupling with carbon fiber (ICF) as carriers to stimulate the biofilms formation for decentralized wastewater treatment was proposed. The typical pollutants removal was accelerated and enhanced (increased by 13.65% for chemical oxygen demand, 19.68% for ammonia nitrogen and 32.66% for phosphate) in ICF compared with the traditional carbon fiber (CF) system. Mechanism explorations indicated that the iron coupling improved the surface properties of carbon fibers and contributed to the attachment and growth of biomass significantly. The components of biomass were changed with increasing proteins proportion in ICF, which was beneficial to the biofilms formation and stability. The microbial community was altered with the enrichment of functional microorganisms (i.e. Pseudomonas and Thauera). Moreover, the microbial metabolic functions (i.e. enzymatic activities and encoding genes) involved in pollutants removal derived from decentralized wastewater were highly expressed in ICF. This work provided an effective strategy to enhance the decentralized wastewater treatment.

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

Resumen Introduccion El sindrome de Cushing ectopico (SCE) es una entidad rara debida a la secrecion de ACTH por tumores extrahipofisarios. Su baja frecuencia dificulta la adquisicion de experiencia en su manejo. El objetivo de este trabajo es describir a los pacientes con SCE atendidos en el servicio de Endocrinologia en un hospital de tercer nivel en un periodo de 15 anos. Metodos Se trata de un estudio retrospectivo de los datos clinicos, bioquimicos y radiologicos, tratamiento recibido, y evolucion de los pacientes con SCE atendidos entre los anos 2000 y 2015. Resultados Se incluyeron 9 pacientes (6 mujeres) con una edad media de 47 anos. El sindrome clinico se desarrollo en un tiempo inferior a 3 meses en todos los casos excepto en uno, y la mayoria presentaba edemas, hiperpigmentacion y/o hipopotasemia. La media del cortisol libre urinario y de la ACTH fue de 2.840 μg/24 h y 204 pg/ml, respectivamente. El origen ectopico se confirmo por la combinacion de pruebas dinamicas no invasivas y estudios radiologicos en la mayoria de los casos. El tumor responsable pudo identificarse en 8 casos y 7 presentaban diseminacion metastasica. El tratamiento primario consistio en cirugia en un caso, cirugia mas terapia sistemica en 3 y quimioterapia en otros 3. En 4 pacientes fue necesaria la suprarrenalectomia bilateral para controlar el hipercortisolismo. Tras un seguimiento medio de 40 meses, 3 habian fallecido, 5 permanecian vivos y en uno se habia perdido el seguimiento. Conclusiones Se confirma que el SCE abarca un amplio espectro de tumores de diferente agresividad y naturaleza. Habitualmente el origen ectopico del sindrome de Cushing puede sospecharse y confirmarse en la mayoria de los casos sin necesidad de pruebas invasivas. Tanto el control del hipercortisolismo como del tumor requieren multiples modalidades terapeuticas, siendo recomendable el manejo multidisciplinar.

Plant-associated bacteria of the genus Xanthomonas cause disease in a wide range of economically important crops. However, their ability to persist in the environment is still poorly understood. Predation by amoebas represents a major selective pressure to bacterial populations in the environment. In this study, we show that the X. citri type 6 secretion system (T6SS) promotes resistance to predation by the soil amoeba Dictyostelium discoideum. We found that an extracytoplasmic function (ECF) sigma factor (EcfK) is required for induction of T6SS genes during interaction with Dictyostelium. EcfK homologues are found in several environmental bacteria in association with a gene encoding a eukaryotic-like Ser/Thr kinase (pknS). Deletion of pknS causes sensitivity to amoeba predation and abolishes induction of T6SS genes. Phosphomimetic mutagenesis of EcfK identified a threonine residue (T51) that renders EcfK constitutively active in standard culture conditions. Moreover, susceptibility of ΔpknS to Dictyostelium predation can be overcome by expression of the constitutively active version EcfKT51E from a multicopy plasmid. Together, these results describe a new regulatory cascade in which PknS functions through activation of EcfK to promote T6SS expression. Our work reveals an important aspect of Xanthomonas physiology that affects its ability to persist in the environment.

Patients with SARS-CoV-2 may be affected by the acute respiratory distress syndrome (ARDS), which has been associated with high mortality rate. As no specific drugs are available for ARDS, mesenchymal stem cells (MSC) seems to be a promising cell therapy due to immunomodulatory effects on reducing and healing inflammation-induced lung and other tissue injuries. The goal of this Phase I clinical trial was to explore the safety and efficacy of bone marrow-derived MSC (BM-MSC) infusions in patients with COVID-19 ARDS. The inclusions criteria were age between 18 to 70 years and PaO2/FiO2≤200mmHg. The BM-MSC infusions were as follow: one to 3 infusions intravenous doses of BM-MSC of 1x10 6 cells/kg; each dose could be administered with an interval between 3 to 7 days. The primary endpoint was safety (adverse events) within 6 hours; cardiac arrest or death within 24 hours post-infusion. The secondary endpoint includes patient survival at 30 days after the first infusion. Six patients were included in the trial and treated with at least one infusion of BM-MSC. The median age was 60,3 years (54 to 69), 5 were male. The median time between the worsening of respiratory distress and the BM-MSC infusion was 10 days (3 to 31 days). The median of PaO2/FiO2 before infusion was 151.86 (127.80-164.44) and median PaCO2 was 63,85 (39 to 117). One patient was treated with 3 MSC doses, two patients with 2 doses and 3 patients one dose. No serious adverse effects were observed within 24 hours post-infusion; only one death was observed following 30 days of cell administration. None of them showed adverse events during BM-MSC infusion. Only one patient showed signs of pulmonary infection one week after first BM-MSC infusion. This patient was at increased risk for infection due to prolonged intubation and a high dose of corticosteroid. Therefore, it was not possible to conclude its association with BM-MSC treatment. Only two patients showed clinical improvement after BM-MSC infusion. Patient 1 had BM-MSC infusion 72 hours after worsening of respiratory parameters, and thorax CT suggested the hypothesis of cryptogenic organizing pneumonia, which led to decision of using methylprednisolone 125mg I.V. for 3 days and carry on with MSC infusion. We observed a decrease in CRP levels from 126 to 67 mg/dL on day 1 after the first infusion, and PaO2/FiO2 ratio improved from 155 to 297 mmHg on day 5. He received the second dose within 7 days interval and by day 11 of the first infusion a new thorax TC showed complete resolution of alveolar consolidation areas in both lungs (Figure 1A and 1B). Patient 2 had BM-MSC administration 11 days after respiratory worsening and also presented improvement of PaO2/FiO2 ratio (148 to 215 mmHg after 2 days of infusion) and had thorax CT images suggesting cryptogenic organizing pneumonia with administration of methylprednisolone 250mg I.V. Nevertheless, the second dose was not administered due to ventilator-associated pneumonia and urinary infection. Four patients showed a non-sustained increase of PaO2/FiO2 ratio, with higher median PCO2 levels of 69,3 mmHg (range, 61,2 to 117) comparing to 39 and 47,3 mmHg of patients 1 and 2, respectively. PCO2 parameter could be a marker to indicate a worse response to MSC treatment, since it could point out chronic phases of COVID-19 disease. The patients died due to COVID-19 complications. No difference in inflammatory markers, such as interleukin 6, C-protein reactive test, procalcitonin, ferritin was observed before and after treatment. Inclusion criteria did not defined interval between respiratory worsening and first BM-MSC infusion. Four patients had chronic phase of COVID-19 without inflammatory markers and hypercapnia. It could be related to severity of pulmonary disease, such as reported in chronic obstructive pulmonary disease. Two patients were discharged after MSC treatment and they received methylprednisolone to treat cryptogenic organizing pneumonia. There are only a few clinical trials and observational studies evaluating the use of high-dose of glucocorticoid for severe COVID-19 pneumonia. Therefore, it is not possible to conclude that use of glucocorticoid has contributed to favorable outcomes. In conclusion, BM-MSC showed to be a secure therapeutic option for severe COVID-19 pneumonia, possibly with superior benefit in acute phases and lower PCO2 levels. Further studies involving a large cohort or randomized controlled trials are warranted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus–mitochondria crosstalk in DENV infected mosquito cells.

Os sistemas de secrecao do tipo VI sao maquinarias de transporte de proteinas para celulas-alvo, que atuam em diversos aspectos da fisiologia bacteriana, incluindo virulencia e competicao com micro-organismos no ambiente. O sistema de secrecao tipo seis (SSVI) de Xanthomonas citri pv. citri (X. citri) e essencial para a resistencia da bacteria a ameba de solo Dictyostelium discoideum. X. citri e um importante fitopatogeno, que causa a doenca cancro citrico em todos os cultivares de citros importantes na atividade agricola brasileira. Assim, e importante identificar os mecanismos moleculares de atuacao do SSVI e as proteinas por ele secretadas (efetores), que conferem o fenotipo de resistencia. O presente trabalho tem por objetivo identificar proteinas que interagem com os componentes do SSVI PAAR e VgrG, proteinas responsaveis pelo recrutamento de efetores para secrecao. Para isso, utilizamos a metodologia de duplo hibrido em leveduras, buscando-se detectar interacoes de proteinas codificadas por biblioteca genomica de X. citri com as proteinas estruturais do SSVI VgrG e PAAR. Paralelamente, estamos realizando testes de interacao direta entre duas proteinas que sao fortes candidatas a efetores com PAAR e VgrG. Interacoes positivas foram obtidas em ensaios de duplo hibrido com biblioteca genomica de X. citri usando-se as proteinas VgrG e PAAR como iscas e o projeto encontra-se atualmente em fase de identificacao destas interacoes por sequenciamento dos clones positivos.

Members of the Xanthomonadales order include several plant pathogens of significant economic and agricultural impact, such as Xanthomonas spp. Type 6 secretion systems (T6SSs) are contractile nanomachines used by many bacterial species to inject protein effectors into target prokaryotic and eukaryotic cells and provide a competitive advantage for bacteria in different environments. Effectors with antibacterial properties include peptidoglycan hydrolases, lipases and phospholipases that break down structural components of the cell envelope, promoting target-cell lysis; and RNases, DNAses, and NADases that affect target-cell metabolism, arresting growth. Effectors with anti-eukaryotic properties are functionally more diverse. The T6SS of Xanthomonas citri is the only example experimentally characterized so far within the Xanthomonadales order and displays anti-eukaryotic function by providing resistance to predation by amoeba. This T6SS is regulated at the transcriptional level by a signaling cascade involving a Ser/Thr kinase and an extracytoplasmic function (ECF) sigma factor. In this review, we performed in silico analyses of 35 genomes of Xanthomonadales and showed that T6SSs are widely distributed and phylogenetically classified into three major groups. In silico predictions identified a series of proteins with known toxic domains as putative T6SS effectors, suggesting that the T6SSs of Xanthomonadales display both anti-prokaryotic and anti-eukaryotic properties depending on the phylogenetic group and bacterial species.

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I-IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors.

Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized molecular machine, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the structural genes of this T4SS in X. citri, is regulated by the conserved global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact-dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri’s aggressive posture when confronted by competitors., Author summary Xanthomonas citri is a member of a family of phytopathogenic bacteria that can cause substantial losses in crops. At different stages of the infection cycle, these cells will encounter other bacterial species with whom they will have to compete for space and nutrients. One mechanism which improves a cell´s chance to survive these encounters is a type IV secretion system that transfers a cocktail of antimicrobial effector proteins into other Gram-negative bacteria in a contact-dependent manner. Here, we show that this system is constitutively produced at a basal level, even during low nutrient conditions, despite representing a significant metabolic burden to the cell. The conserved global regulator, CsrA, provides a constant, nutrient-independent, repression on the production of T4SS components, thereby holding production costs to a minimum while at the same time ensuring X. citri’s competitiveness during encounters with bacterial rivals.

Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.

Studies on species distribution models evaluate the reliability and discrimination capacity of the models, while the accuracy of the spatial component of the data is often disregarded. The objective of our work was to design and validate an index, the Georeferencing Reliability Index (GRI), to measure and compare quality of different databases containing presence data of Aotus species. The database of A. lemurinus showed the highest georeferencing accuracy (GRI = 0.608) and that of A. zonalis the lowest (GRI = 0.167). Results indicated that there is poor-quality spatial information data for Aotus spp. Therefore, the distribution areas of all species should be more accurately established to evaluate whether they are at conservation risk. In addition, the index can be used to select the most accurate spatial data for constructing a species distribution model. Finally, we strongly believe that the GRI may provide accurate, up-to-date information essential for wildlife management programs.

Wolbachia sp. has colonized over 70% of insect species, successfully manipulating host fertility, protein expression, lifespan, and metabolism. Understanding and engineering the biochemistry and physiology of Wolbachia holds great promise for insect vector‐borne disease eradication. Wolbachia is cultured in cell lines, which have long duplication times and are difficult to manipulate and study. The yeast strain Saccharomyces cerevisiae W303 was used successfully as an artificial host for Wolbachia wAlbB. As compared to controls, infected yeast lost viability early, probably as a result of an abnormally high mitochondrial oxidative phosphorylation activity observed at late stages of growth. No respiratory chain proteins from Wolbachia were detected, while several Wolbachia F1F0‐ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.

Ectopic Cushing's syndrome (ECS) is a rare condition caused by ACTH secretion by extrapituitary tumors. Its low frequency makes it difficult to acquire experience in its management. The aim of this study was to describe patients with ECS seen at the endocrinology department of a tertiary hospital over 15 years.This was a retrospective study of the clinical, biochemical and radiographic data, treatment, and course of patients with ECS seen from 2000 to 2015.Nine patients (6 of them female) with a mean age of 47 years were included in the study. The clinical syndrome developed in less than 3 months in all cases but one, and most patients also had edema, hyperpigmentation and/or hypokalemia. Mean urinary free cortisol and ACTH levels were 2,840μg/24h and 204pg/mL respectively. The ectopic origin was confirmed by a combination of dynamic non-invasive tests and radiographic studies in most cases. The tumor responsible could be identified in 8 cases, and 7 patients had metastatic dissemination. Primary treatment was surgery in one patient, surgery combined with systemic therapy in 3, and chemotherapy in the other 3 patients. Bilateral adrenalectomy was required in 4 patients to control hypercortisolism. After a mean follow-up of 40 months, 3 patients died, 5 were still alive, and one had been lost to follow-up.Our study confirms that ECS covers a wide spectrum of tumors of different aggressiveness and nature. The ectopic origin of Cushing's syndrome can usually, be suspected and confirmed in most cases without the need for invasive tests. Control of both hypercortisolism and the tumor requires multiple treatment modalities, and multidisciplinary management is recommended.

Plant-associated bacteria of the genus Xanthomonas cause disease in a wide range of economically important crops. However, their ability to persist in the environment is still poorly understood. Predation by amoebas represents a major selective pressure to bacterial populations in the environment. In this study, we show that the X. citri type 6 secretion system (T6SS) promotes resistance to predation by the soil amoeba Dictyostelium discoideum. We found that an extracytoplasmic function (ECF) sigma factor (EcfK) is required for induction of T6SS genes during interaction with Dictyostelium. EcfK homologues are found in several environmental bacteria in association with a gene encoding a eukaryotic-like Ser/Thr kinase (pknS). Deletion of pknS causes sensitivity to amoeba predation and abolishes induction of T6SS genes. Phosphomimetic mutagenesis of EcfK identified a threonine residue (T51) that renders EcfK constitutively active in standard culture conditions. Moreover, susceptibility of ΔpknS to Dictyostelium predation can be overcome by expression of the constitutively active version EcfK

Background Autologous peripheral blood hematopoietic stem cell (PBSC) collection efficiency (CE) is reportedly affected by the patient's blood properties; however, studies to identify factors correlated with CE have shown inconsistent results. Additionally, variables such as stem cell graft granulocyte content and patient age, sex, and underlying disease, may be associated with hematopietic stem cell (HSC) infusion-related adverse reactions. In this study, we evaluated the correlation of preleukapheresis PB granulocyte count and PBSC harvest variables with CD34+ collection yield and efficiency, and thawed HSC infusion side effect occurrence. Patients and methods We evaluated data from 361 patients who had undergone autologous PBSC transplant. Large volume leukapheresis was the method for PBSC collection. Complete Blood Count and CD34+ cell enumeration were performed in the preapheresis PB and the apheresis product sample. The PBSC grafts were submitted to non-controlled rate freezing after addition of 5% DMSO plus 6% hidroxyethylstarch as a cryoprotectant solution. The cryopreserved graft was thawed in a 37°C water bath and then infused without further manipulation. Results The CD34+ yield was associated with preapheresis PB CD34+ count and immature granulocyte count. The PBSC CE was negatively correlated with preapheresis white blood cell (WBC), immature granulocyte and granulocyte count. The leukapheresis product total nucleated cell (TNC) and granulocyte content was correlated with the thawed graft infusion side effect occurrence. Conclusion This study has shown that preapheresis PB WBC and granulocyte counts were associated with leukapheresis CE. Additionally, the leukapheresis product TNC and granulocyte content was correlated with thawed graft infusion side effect occurrence.

Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments, infecting poultry and eggs. Bacteria in biofilms are difficult to eradicate compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated the role of ihfA and ihfB in biofilm formation by S. enterica Enteritidis by employing different microbiology techniques. Our data indicate that ihf mutant strains are impaired in biofilm formation, showing a reduction in matrix formation and a decrease in viability and metabolic activity. Phenotypic analysis also showed that deletion of ihf causes a deficiency in curli fimbriae expression, cellulose production and pellicle formation. These results show that integration host factor has an important regulatory role in biofilm formation by S. enterica Enteritidis.

Ubiquinone derivatives modulate the mammalian mitochondrial Permeability Transition Pore (PTP). Yeast mitochondria harbor a similar structure: the respiration- and ATP-induced Saccharomyces cerevisiae Mitochondrial Unselective Channel ( Sc MUC). Here we show that decylubiquinone, a well-characterized inhibitor of the PTP, suppresses Sc MUC opening in diverse strains and independently of respiratory chain modulation or redox-state. We also found that naturally occurring derivatives such as hexaprenyl and decaprenyl ubiquinones lacked effects on the Sc MUC. The PTP-inactive ubiquinone 5 (Ub5) promoted the Sc MUC-independent activation of the respiratory chain in most strains tested. In an industrial strain however, Ub5 blocked the protection elicited by dUb. The results indicate the presence of a ubiquinone-binding site in the Sc MUC.

First results of a multi-disciplinary hyporheic monitoring study are presented from the newly established Steinlach Test Site in Southern Germany. The site is located in a bend of the River Steinlach (mean discharge of 1.8 m³/s) underlain by an alluvial sandy gravel aquifer connected to the stream. The overall objective is a better understanding of hyporheic exchange processes at the site and their interrelations with microbial community dynamics and biochemical reactions at the stream–groundwater interface. The present paper focuses on the distribution of lateral hyporheic exchange fluxes and their associated travel times at the Steinlach Test Site. Water level dynamics in various piezometers correspond to the different domains of hydraulic conductivity in the shallow aquifer and confirms hyporheic exchange of infiltrated stream water across the test site. Hydrochemical compositions as well as increased damping of continuous time series of electrical conductivity (EC) and temperature at the respective piezometers confirmed the inferred distribution of hyporheic flowpaths. Mean travel times ranging from 0.5 days close to the stream to more than 8 days in the upstream part of the test site could be estimated from deconvolution of EC and δ18O–H2O data. The travel times agree well with the presumed flowpaths. Mg/Ca ratios as well as model fits to the EC and δ18O data indicate the presence of an additional water component in the western part of the test site which most likely consists of hillslope water or groundwater. Based on the mean travel times, the total lateral hyporheic exchange flux at the site was estimated to be of the order of 1–2 L/s.

Oophorectomy in adult rats affected cardiac mitochondrial function. Progression of mitochondrial alterations was assessed at one, two and three months after surgery: at one month, very slight changes were observed, which increased at two and three months. Gradual effects included decrease in the rates of oxygen consumption and in respiratory uncoupling in the presence of complex I substrates, as well as compromised Ca2+buffering ability. Malondialdehyde concentration increased, whereas the ROS-detoxifying enzyme Mn2+superoxide dismutase (MnSOD) and aconitase lost activity. In the mitochondrial respiratory chain, the concentration and activity of complex I and complex IV decreased. Among other mitochondrial enzymes and transporters, adenine nucleotide carrier and glutaminase decreased. 2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase also decreased. Data strongly suggest that in the female rat heart, estrogen depletion leads to progressive, severe mitochondrial dysfunction.