A Ranking Tool for "Category Killer" Microbial Biobanks.

Microbial biobanks preserve and provide microbial bioresources for research, training, and quality control purposes. They ensure the conservation of biodiversity, contribute to taxonomical research, and support scientific advancements. Microbial biobanks can cover a wide range of phylogenetic and metabolic diversity ("category killers") or focus on specific taxonomic, thematic, or disease areas. The strategic decisions about strain selection for certain applications or for the biobank culling necessitate a method to support prioritization and selection. Here, we propose an unbiased scoring approach based on objective parameters to assess, categorize, and assign priorities among samples in stock in a microbial biobank. We describe the concept of this ranking tool and its application to identify high-priority strains for whole genome sequencing with two main goals: (i) genomic characterization of quality control, reference, and type strains; (ii) genome mining for the discovery of natural products, bioactive and antimicrobial molecules, with focus on human diseases. The general concept of the tool can be useful to any biobank and for any ranking or culling needs.

Síndrome de arlequim idiopática: sucesso terapêutico com toxina botulínica / Idiopathic harlequin syndrome: successful therapy with botulium toxin

A síndrome de arlequim é uma rara desordem autonômica que se caracteriza por anidrose e falta de rubor unilateral da face, podendo acometer as regiões cervical e torácica. De forma paradoxal, há rubor e sudorese compensatórios no lado contralateral à alteração. É idiopática na maioria dos casos, mas pode ser congênita, secundária a lesões estruturais e à iatrogenia pós-cirúrgica. O tratamento é direcionado ao fator causal. Descreve-se caso de paciente com diagnóstico de síndrome de arlequim idiopática, sendo realizada aplicação de toxina botulínica na hemiface acometida pelos sintomas compensatórios com boa resposta terapêutica

Harlequin syndrome is a rare autonomic disorder characterized by anhidrosis and lack of unilateral flushing of the face, which may affect the cervical and thoracic regions. Paradoxically, there is compensatory flushing and sweating on the contralateral side to the alteration. It is idiopathic in most cases, but it can be congenital or secondary to structural or post-surgical iatrogenic lesions. Treatment is directed at the causative factor. We describe the case of a patient with a diagnosis of idiopathic Harlequin Syndrome with botulinum toxin application in the hemiface affected by compensatory symptoms with good therapeutic response.

Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution.

Bacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with full-length IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states.

Shigella impairs human T lymphocyte responsiveness by hijacking actin cytoskeleton dynamics and T cell receptor vesicular trafficking.

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.

Shigella-mediated oxygen depletion is essential for intestinal mucosa colonization.

Pathogenic enterobacteria face various oxygen (O2) levels during intestinal colonization from the O2-deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O2 in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O2 is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

Upper gastrointestinal alterations in kidney transplant candidates / Alterações digestivas altas em candidatos a transplante renal

ABSTRACT Introduction: The incidence of gastrointestinal disorders among patients with chronic kidney disease (CKD) is high, despite the lack of a good correlation between endoscopic findings and symptoms. Many services thus perform upper gastrointestinal (UGI) endoscopy on kidney transplant candidates. Objectives: This study aims to describe the alterations seen on the upper endoscopies of 96 kidney-transplant candidates seen from 2014 to 2015. Methods: Ninety-six CKD patients underwent upper endoscopic examination as part of the preparation to receive kidney grafts. The data collected from the patients' medical records were charted on Microsoft Office Excel 2016 and presented descriptively. Mean values, medians, interquartile ranges and 95% confidence intervals of the clinic and epidemiological variables were calculated. Possible associations between endoscopic findings and infection by H. pylori were studied. Results: Males accounted for 54.17% of the 96 patients included in the study. Median age and time on dialysis were 50 years and 50 months, respectively. The most frequent upper endoscopy finding was enanthematous pangastritis (57.30%), followed by erosive esophagitis (30.20%). Gastric intestinal metaplasia and peptic ulcer were found in 8.33% and 7.30% of the patients, respectively. H. pylori tests were positive in 49 patients, and H. pylori infection was correlated only with non-erosive esophagitis (P = 0.046). Conclusion: Abnormal upper endoscopy findings were detected in all studied patients. This study suggested that upper endoscopy is a valid procedure for kidney transplant candidates. However, prospective studies are needed to shed more light on this matter.

RESUMO Introdução: A incidência de doenças gastrointestinais altas em pacientes com doença renal crônica é elevada, porém não há boa correlação entre achados endoscópicos e sintomas. Assim, muitos serviços preconizam a realização de Endoscopia Digestiva Alta (EDA) nos candidatos a transplante renal. Objetivos: Descrever alterações endoscópicas presentes em 96 candidatos a transplante renal no período de 2014 a 2015. Métodos: Noventa e seis pacientes renais crônicos submetidos à EDA como preparo para transplante renal. Prontuários médicos dos pacientes foram revisados, os dados tabulados no programa Microsoft Office Excel 2016 e apresentados de maneira descritiva. Calculou-se média, mediana, intervalo interquartílico e intervalo de confiança de 95% das variáveis utilizadas. Alterações endoscópicas foram apresentadas quanto ao número, intervalo de confiança e valor de P, e correlacionadas com a presença ou ausência de infecção por Helicobacter pylori. Resultados: Dos 96 pacientes, 54,17% eram homens e 45,83% mulheres. As medianas de idade e tempo em diálise foram 50 anos e 50 meses, respectivamente. O achado mais comum na EDA foi pangastrite enantematosa (57,30%), seguida de esofagite erosiva (30,20%). Metaplasia intestinal gástrica e úlcera péptica foram encontradas em 8,33% e 7,30% dos pacientes, respectivamente. Pesquisa para H. pylori foi positiva em 49 pacientes, e somente houve correlação entre infecção por H. pylori e esofagite não erosiva (P = 0,046). Conclusão: Afecções gastrointestinais foram detectadas em todos os pacientes estudados. Os achados deste estudo sugerem que a realização de EDA em candidatos a receber transplante renal é desejável. Entretanto, estudos prospectivos são necessários para responder a esta questão.

Upper gastrointestinal alterations in kidney transplant candidates.

INTRODUCTION: The incidence of gastrointestinal disorders among patients with chronic kidney disease (CKD) is high, despite the lack of a good correlation between endoscopic findings and symptoms. Many services thus perform upper gastrointestinal (UGI) endoscopy on kidney transplant candidates. OBJECTIVES: This study aims to describe the alterations seen on the upper endoscopies of 96 kidney-transplant candidates seen from 2014 to 2015. METHODS: Ninety-six CKD patients underwent upper endoscopic examination as part of the preparation to receive kidney grafts. The data collected from the patients' medical records were charted on Microsoft Office Excel 2016 and presented descriptively. Mean values, medians, interquartile ranges and 95% confidence intervals of the clinic and epidemiological variables were calculated. Possible associations between endoscopic findings and infection by H. pylori were studied. RESULTS: Males accounted for 54.17% of the 96 patients included in the study. Median age and time on dialysis were 50 years and 50 months, respectively. The most frequent upper endoscopy finding was enanthematous pangastritis (57.30%), followed by erosive esophagitis (30.20%). Gastric intestinal metaplasia and peptic ulcer were found in 8.33% and 7.30% of the patients, respectively. H. pylori tests were positive in 49 patients, and H. pylori infection was correlated only with non-erosive esophagitis (P = 0.046). CONCLUSION: Abnormal upper endoscopy findings were detected in all studied patients. This study suggested that upper endoscopy is a valid procedure for kidney transplant candidates. However, prospective studies are needed to shed more light on this matter.

Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using ß-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes.

The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.

Protein-templated biomimetic silica nanoparticles.

Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250-380 nm) and were more monodisperse than those lacking the proteic core (∼700-1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g(-1) of particles.

Spatial organization and stoichiometry of N-terminal domain-mediated glycosyltransferase complexes in Golgi membranes determined by fret microscopy.

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.

An insect growth inhibitor--lufenuron--enhances albendazole activity against hydatid cyst.

The aim of this work was to evaluate the potential of lufenuron, a benzylphenylurea with ability to interfere with the formation of insect exoskeleton, as a therapeutic drug for larval echinococcosis (hydatid disease). For this purpose lufenuron, alone or in combination with albendazole, was administered to CD1 mice bearing Echinococcus granulosus hydatid cysts in the peritoneal cavity. Neither of the drugs alone was able to exert parasiticidal effects. However, in combination with albendazole, lufenuron reduced the growth of cysts (30-40% in cyst diameter respect to control, p<0.05). This effect was associated with ultrastructural alterations of the hydatid cyst wall and a reduction of the content of myo-inositol-hexakisphosphate, the major component of the electron dense granules of the laminated layer. Overall, this work provides evidence that lufenuron could represent a useful compound for the use in chemotherapy against larval echinococcosis, by enhancing albendazole parasiticidal activity.

Cellular and molecular biology of glycosphingolipid glycosylation.

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.

Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate.

Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signalling mediators, based on their high-affinity PPARgamma (peroxisome-proliferator-activated receptor gamma) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO2 (cholesteryl-nitrolinoleate). CLNO2 levels were found to increase approximately 20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-gamma; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the (*)NO (nitric oxide) inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrow-derived cells) inflammatory responses were suppressed when activated in the presence of CLNO2 or LNO2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARgamma and *NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-kappaB (nuclear factor kappaB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.

Study of the relationship between Aedes (Stegomyia) aegypti egg and adult densities, dengue fever and climate in Mirassol, state of São Paulo, Brazil.

The purpose of this study was to examine the relationship between Aedes aegypti egg and adult density indices, dengue fever and climate in Mirassol, state of São Paulo, Brazil, between November 2004-November 2005. Weekly collections of adults and eggs were made using, respectively, manual aspirators and oviposition traps that produced four entomological indices (positivity and average of females and eggs). Weekly incidence coefficients were calculated based on dengue cases. Each week, the data obtained from entomological indices were related to each other, dengue, and climate variables. The first index to show an association with dengue transmission was the female average, followed by female positivity and egg average. Egg positivity did not show a relationship with risk for dengue, but was sensitive to identifying the presence of the vector, principally in dry seasons. The relationship between climatic factors, the vector and the disease found in this study can be widely employed in planning and undertaking dengue surveillance and control activities, but it is a tool that has not been considered by the authorities responsible for controlling the disease. In fact, this relationship permits the use of information about climate for early detection of epidemics and for establishing more effective prevention strategies than currently exist.

Study of the relationship between Aedes (Stegomyia) aegypti egg and adult densities, dengue fever and climate in Mirassol, state of São Paulo, Brazil

The purpose of this study was to examine the relationship between Aedes aegypti egg and adult density indices, dengue fever and climate in Mirassol, state of São Paulo, Brazil, between November 2004-November 2005. Weekly collections of adults and eggs were made using, respectively, manual aspirators and oviposition traps that produced four entomological indices (positivity and average of females and eggs). Weekly incidence coefficients were calculated based on dengue cases. Each week, the data obtained from entomological indices were related to each other, dengue, and climate variables. The first index to show an association with dengue transmission was the female average, followed by female positivity and egg average. Egg positivity did not show a relationship with risk for dengue, but was sensitive to identifying the presence of the vector, principally in dry seasons. The relationship between climatic factors, the vector and the disease found in this study can be widely employed in planning and undertaking dengue surveillance and control activities, but it is a tool that has not been considered by the authorities responsible for controlling the disease. In fact, this relationship permits the use of information about climate for early detection of epidemics and for establishing more effective prevention strategies than currently exist.

Nitroalkenes: synthesis, characterization, and effects on macrophage activation.

Nitroalkenes derivatives of free as well as esterified unsaturated fatty acids are present in human plasma and tissue, representing novel pluripotent cell signaling mediators. Lipid nitration occurs in response to pro-inflammatory stimuli as an adaptative mechanism to downregulate inflammatory responses. This chapter first discusses the generation of nitroalkenes during macrophage activation following chemical and biological characterization. In particular, it describes procedures for (a) synthesizing and characterizing esterified (cholesteryl-nitrolinoleate, CLNO2) as well as free (nitroarachidonate, AANO2) nitroalkenes, (b) determining nitration of cholesteryl linoleic acid during macrophage activation by inflammatory stimuli, (c) examining the modulatory effects of nitroalkenes on the expression of inducible enzymes by activated macrophages, and (d) discussing the signaling pathways involved in nitroalkene-mediated anti-inflammatory actions.

Calsenilin and CALP interact with the cytoplasmic tail of UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase.

GalT2 (UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase) is a Golgi-resident type II membrane protein that participates in the synthesis of glycosphingolipids. The molecular determinants for traffic and localization of this and other glycosyltransferases are still poorly characterized. Considering the possibility that interactions with other proteins may influence these processes, in the present study we carried out a yeast two-hybrid screening using elements of the N-terminal domain of GalT2 as bait. In this screening, we identified calsenilin and its close homologue CALP (calsenilin-like protein), both members of the recoverin-NCS (neuronal calcium sensor) family of calcium-binding proteins. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in CHO (Chinese-hamster ovary)-K1 cells. The expression of CALP or calsenilin affect Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 (CMP-NeuAc:GM3 sialyltransferase) and GalNAcT (UDP-GalNAc:lactosylceramide/GM3/GD3 beta1-4 N-acetylgalactosaminyltransferase), by redistributing them from the Golgi to the ER (endoplasmic reticulum), whereas the localization of the VSV-G (G-protein of the vesicular stomatitis virus) or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases.