A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

A Digital Image-Based Phenotyping Platform for Analyzing Root Shape Attributes in Carrot.

Root shape in carrot (Daucus carota subsp. sativus), which ranges from long and tapered to short and blunt, has been used for at least several centuries to classify carrot cultivars. The subjectivity involved in determining market class hinders the establishment of metric-based standards and is ill-suited to dissecting the genetic basis of such quantitative phenotypes. Advances in digital image acquisition and analysis has enabled new methods for quantifying sizes of plant structures and shapes, but in order to dissect the genetic control of the shape features that define market class in carrot, a tool is required that quantifies the specific shape features used by humans in distinguishing between classes. This study reports the construction and demonstration of the first such platform, which facilitates rapid phenotyping of traits that are measurable by hand, such as length and width, as well as principal component analysis (PCA) of the root contour and its curvature. This latter approach is of particular interest, as it enabled the detection of a novel and significant quantitative trait, defined here as root fill, which accounts for 85% of the variation in root shape. Curvature analysis was demonstrated to be an effective method for precise measurement of the broadness of the carrot shoulder, and degree of tip fill; the first principal component of the respective curvature profiles captured 87% and 84% of the total variance. This platform's performance was validated in two experimental panels. First, a diverse, global collection of germplasm was used to assess its capacity to identify market classes through clustering analysis. Second, a diallel mating design between inbred breeding lines of differing market classes was used to estimate the heritability of the key phenotypes that define market class, which revealed significant variation in the narrow-sense heritability of size and shape traits, ranging from 0.14 for total root size, to 0.84 for aspect ratio. These results demonstrate the value of high-throughput digital phenotyping in characterizing the genetic control of complex quantitative phenotypes.

Molecular diagnosis of meningitis and meningoencephalitis with an automated real-time multiplex polymerase chain reaction in a tertiary reference complex in Medellín, Colombia.

Meningoencephalitis is a syndrome of multiple etiologies associated with important morbidity and mortality. It may be caused by various infectious agents (viruses, bacteria, parasites and fungi). Establishing the etiology of meningoencephalitis is crucial for early and specific treatment. Molecular assays such as the multiplex polymerase chain reaction (PCR) offer an alternative in diagnosing central nervous system infections. This study aimed to describe the performance of an automated multiplex molecular test from patients with suspected meningitis and meningoencephalitis in a tertiary referral complex in Medellin, Colombia. Thus, a prospective study was performed in 638 cerebrospinal fluid samples from January 2017 to July 2019. Molecular detections were carried out by means of the FilmArray® Meningitis/Encephalitis (M/E) Panel from bioMérieux, France, and by conventional tests. Univariate analyses for microbiological and demographic characteristics were performed. Accuracy of the bacterial/fungal PCR assay compared to cultures was also performed. Among patients, 57.7% were male, the median age was 24 (IQR: 6 - 47) years old. The overall positivity was 15.2% (97 detections) and viruses were detected in 45.5% of the samples, bacteria in 43.5% and fungi in 10.8%. The most frequent etiological agents were: Streptococcus pneumoniae (16%), Cryptococcus neoformans/gatti (11.3%) and Herpes simplex virus (10.3%). Four double detections were found. Almost half of positive detections were in patients under 15 years old. This molecular approach is reliable and easily implantable into a laboratory routine, increasing the capacity of detection of bacterial and viral causative agents of meningitis, possibly playing a relevant role in the clinical context.

Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of α-ketoglutarate.

To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

Diagnóstico molecular de tuberculosis extrapulmonar y sensibilidad a rifampicina con un método automatizado en tiempo real / Molecular diagnosis of extrapulmonary tuberculosis and sensitivity to rifampicin with an automated real-time method

Introducción. La tuberculosis continúa siendo un problema de salud pública mundial, y la forma extrapulmonar representa entre 10 y 20 % del total de casos en personas inmunocompetentes, porcentaje que se incrementa en los portadores del virus de la inmunodeficiencia humana (HIV). Su diagnóstico es difícil con los métodos convencionales por la naturaleza paucibacilar de las muestras. La prueba Xpert ® MTB/RIF ha significado un importante avance en la detección molecular de Mycobacterium tuberculosis y hoy se utiliza en una variedad de muestras clínicas obtenidas de sitios diferentes a las vías respiratorias. Objetivo. Determinar la utilidad de la prueba Xpert ® MTB/RIF en la detección de M. tuberculosis y la sensibilidad a la rifampicina en pacientes con sospecha de tuberculosis extrapulmonar, atendidos en el Hospital Universitario de San Vicente Fundación de Medellín entre 2013 y 2014. Materiales y métodos. Se hizo un estudio descriptivo de corte transversal retrospectivo y prospectivo en 372 muestras consecutivas provenientes de 301 pacientes con sospecha de tuberculosis extrapulmonar, las cuales se analizaron mediante baciloscopia, cultivo en medio de Ogawa-Kudoh y prueba molecular Xpert ® MTB/RIF. Resultados. De los pacientes estudiados, 182 eran hombres (60 %) y la condición de base más frecuentemente diagnosticada fue la infección por HIV. Al tomar como referencia el cultivo, la sensibilidad y la especificidad general de la prueba molecular fueron de 94 % (IC 95% 83-100) y 97 % (IC 95% 95-99), respectivamente, y para la baciloscopia, fueron de 38,71 % (IC 95% 19-57) y 100 % (IC 95% 99-100), respectivamente. En el análisis estratificado por muestras se encontraron sensibilidades mayores de 75 %; 37 aislamientos fueron sensibles a la rifampicina y uno fue resistente. Conclusión. La prueba Xpert ® MTB/RIF tuvo un buen desempeño en muestras de diferentes tejidos y líquidos, y constituye un avance significativo como apoyo para el diagnóstico de la tuberculosis extrapulmonar en términos de tiempo y porcentaje de positividad.

Introduction: Tuberculosis continues to be a global public health problem, the extrapulmonary form being estimated to occur in 10-20% of immunocompetent individuals, increasing in patients who are carriers of the human immunodeficiency virus (HIV); its diagnosis is difficult with conventional methods due to the paucibacillary nature of samples. The Xpert ® MTB/RIF test represents an important development in the molecular detection of Mycobacterium tuberculosis and has been used with a variety of non-respiratory clinical samples. Objective: To determine the effectiveness of Xpert ® MTB/RIF in the detection of M. tuberculosis and sensitivity to rifampicin in patients with suspected extrapulmonary tuberculosis attending Hospital Universitario de San Vicente Fundación in Medellín in 2013-2014. Materials and methods: This was a descriptive, cross-sectional ambispective study of 372 consecutive samples from 301 patients with suspected extrapulmonary tuberculosis, who were subjected to bacilloscopy, followed by culture in Ogawa Kudoh and the Xpert ® MTB/RIF molecular test. Results: The most frequent base diagnosis (60%) for the 182 patients was infection with HIV. Using the culture as reference, the sensitivity and general specificity of the molecular test was 94% (95% CI: 83-100) and 97% (95% CI: 95-99), respectively; for bacilloscopy it was 38.71(95% CI: 19-57) and 100% (95% CI: 99-100), respectively. Sensitivities higher than 75% were found in analyses stratified by samples. Thirty-seven of the isolates were sensitive and one resistant to rifampicin. Conclusion: Xpert ® MTB/RIF performed well in samples from different tissues and liquids, representing a significant advance in support of extrapulmonary tuberculosis diagnosis in terms of time and percentage positivity.

Molecular diagnosis of extrapulmonary tuberculosis and sensitivity to rifampicin with an automated real-time method.

INTRODUCTION: Tuberculosis continues to be a global public health problem, the extrapulmonary form being estimated to occur in 10-20% of immunocompetent individuals, increasing in patients who are carriers of the human immunodeficiency virus (HIV); its diagnosis is difficult with conventional methods due to the paucibacillary nature of samples. The Xpert® MTB/RIF test represents an important development in the molecular detection of Mycobacterium tuberculosis and has been used with a variety of non-respiratory clinical samples.  OBJECTIVE: To determine the effectiveness of Xpert® MTB/RIF in the detection of M. tuberculosis and sensitivity to rifampicin in patients with suspected extrapulmonary tuberculosis attending Hospital Universitario de San Vicente Fundación in Medellín in 2013-2014.  MATERIALS AND METHODS: This was a descriptive, cross-sectional ambispective study of 372 consecutive samples from 301 patients with suspected extrapulmonary tuberculosis, who were subjected to bacilloscopy, followed by culture in Ogawa Kudoh and the Xpert® MTB/RIF molecular test.  RESULTS: The most frequent base diagnosis (60%) for the 182 patients was infection with HIV. Using the culture as reference, the sensitivity and general specificity of the molecular test was 94% (95% CI: 83-100) and 97% (95% CI: 95-99), respectively; for bacilloscopy it was 38.71(95% CI: 19-57) and 100% (95% CI: 99-100), respectively. Sensitivities higher than 75% were found in analyses stratified by samples. Thirty-seven of the isolates were sensitive and one resistant to rifampicin.  CONCLUSION: Xpert® MTB/RIF performed well in samples from different tissues and liquids, representing a significant advance in support of extrapulmonary tuberculosis diagnosis in terms of time and percentage positivity.

The taurine transporter substrate guanidinoethyl sulfonate mimics the action of taurine on long-term synaptic potentiation.

Taurine is especially abundant in rodent brain where it appears to be involved in osmoregulation and synaptic plasticity mechanisms. The demonstration of a physiological role for taurine has been hampered by the difficulty in modifying taurine levels in most tissues, including the brain. We used an experimental strategy to reduce taurine levels, involving treatment with guanidinoethyl sulfonate (GES), a structural analogue of taurine that, among other properties, acts as a competitive inhibitor of taurine transport. GES delivered in the drinking water of rats for 1 month effectively reduced taurine levels in brain structures (hippocampus, cerebellum and cortex) and outside the brain (heart, muscle, kidney, liver and plasma) by between 50 and 80 %, depending on the tissue. This partial taurine depletion did not affect either basal synaptic transmission or the late phase of long-term potentiation (late-LTP) in hippocampal slices. In vivo microdialysis studies in the hippocampus revealed that GES treatment reduced extracellular taurine levels and the magnitude of taurine released in response to the application of either N-methyl-D-aspartate (NMDA) or a hypoosmotic solution, without affecting release mechanisms. Finally, we demonstrated in hippocampal slices that a brief GES application can mimic taurine action on the conversion of a decremental LTP into a perdurable late-LTP, concluding that GES might replace taurine function in some mechanisms such as those implicated in synaptic plasticity.

Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression.

BACKGROUND: Genetic models have been developed in divergent branches of the class Alphaproteobacteria to help answer a wide spectrum of questions regarding bacterial physiology. For example, Sinorhizobium meliloti serves as a useful representative for investigating rhizobia-plant symbiosis and nitrogen fixation, Caulobacter crescentus for studying cell cycle regulation and organelle biogenesis, and Zymomonas mobilis for assessing the potentials of metabolic engineering and biofuel production. A tightly regulated promoter that enables titratable expression of a cloned gene in these different models is highly desirable, as it can facilitate observation of phenotypes that would otherwise be obfuscated by leaky expression. RESULTS: We compared the functionality of four promoter regions in S. meliloti (P(araA), P(tauA), P(rhaR), and P(melA)) by constructing strains carrying fusions to the uidA reporter in their genomes and measuring beta-glucuronidase activities when they were induced by arabinose, taurine, rhamnose, or melibiose. P(tauA) was chosen for further study because it, and, to a lesser extent, P(melA), exhibited characteristics suitable for efficient modulation of gene expression. The levels of expression from P(tauA) depended on the concentrations of taurine, in both complex and defined media, in S. meliloti as well as C. crescentus and Z. mobilis. Moreover, our analysis indicated that TauR, TauC, and TauY are each necessary for taurine catabolism and substantiated their designated roles as a transcriptional activator, the permease component of an ABC transporter, and a major subunit of the taurine dehydrogenase, respectively. Finally, we demonstrated that P(tauA) can be used to deplete essential cellular factors in S. meliloti, such as the PleC histidine kinase and TatB, a component of the twin-arginine transport machinery. CONCLUSIONS: The P(tauA) promoter of S. meliloti can control gene expression with a relatively inexpensive and permeable inducer, taurine, in diverse alpha-proteobacteria. Regulated expression of the same gene in different hosts can be achieved by placing both tauR and P(tauA) on appropriate vectors, thus facilitating inspection of conservation of gene function across species.

Cooperation of taurine uptake and dopamine D1 receptor activation facilitates the induction of protein synthesis-dependent late LTP.

Co-activation of NMDA and dopamine receptors is required for the induction of the late phase of LTP (L-LTP) that is dependent on new protein synthesis. Other neuromodulatory substances may also contribute to this process. Here, we examined whether taurine is one of the neuromodulators contributing to L-LTP induction, since it is known that taurine uptake induces a long-lasting synaptic potentiation dependent on protein synthesis, and taurine uptake inhibition blocks L-LTP induced by tetanization. Experiments were conducted using rat hippocampal slices where field synaptic potentials were evoked and recorded in CA3-CA1 synapses. Taurine (1 mM) applied 10 min before a high frequency stimulation (HFS) train converted a transitory early-LTP (E-LTP) into an L-LTP dependent on protein synthesis. This taurine effect was blocked by a taurine uptake inhibitor. A facilitation of L-LTP induction was also obtained by pre-application of SKF38393, a D1/D5 dopamine receptor (D1R) agonist. In this case, LTP facilitation was not affected by the taurine uptake inhibitor. Nevertheless, when taurine and SKF38393 were simultaneously pre-applied at a concentration that individually did not modify E-LTP, they produced a synergistic mechanism that facilitated the induction of L-LTP with a sole HFS train. This facilitation of L-LTP was blocked by inhibiting either taurine uptake or D1R activation. Taurine and SKF38393 activated different signaling pathways to transform E-LTP into L-LTP. Taurine-induced L-LTP facilitation required MAPK activation, while D1R-agonist-induced facilitation depended mainly on PKA activation and partially on MAPK activation. On the other hand, the synergistic mechanisms induced by the cooperative action of taurine and SKF38393 were impaired by inhibitors against MAPK, PKA and PI3-K. This pharmacological profile resembles that displayed by L-LTP induced by three HFS trains at 10-min intervals. These results indicate that taurine uptake is necessary and cooperates with other neurotransmitter systems in the induction of L-LTP.

Application of a low-cost web-based simulation to improve students' practical skills in medical education.

BACKGROUND: Practical sessions in undergraduate medical education are often costly and have to face constraints in terms of available laboratory time and practice materials (e.g. blood samples from animals). This makes it difficult to increase the time each student spends at the laboratory. We consider that it would be possible to improve the effectiveness of the laboratory time by providing the students with computer-based simulations for prior rehearsal. However, this approach still presents issues in terms of development costs and distribution to the students. OBJECTIVE: This study investigates the employment of low-cost simulation to allow medical students to rehearse practical exercises through a web-based e-learning environment. The aim is to maximize the efficiency of laboratory time and resources allocated by letting students become familiarized with the equipment and the procedures before they attend a laboratory session, but without requiring large-scale investment. Moreover, students can access the simulation via the Internet and rehearse at their own pace. We have studied the effects of such a simulation in terms of impact on the laboratory session, learning outcomes and student satisfaction. METHODS: We created a simulation that covers the steps of a practical exercise in a Physiology course (measuring hematocrit in a blood sample). An experimental group (EG, n=66) played the simulation 1 week before the laboratory session. A control group (CG, n=77) attended the laboratory session without playing the simulation. After the session, all students completed a survey about their perception of the difficulty of the exercise on a scale of 1-10 and the HCT final value that they obtained. The students in the EG also completed a survey about their satisfaction with the experience. RESULTS: After the laboratory session, the perceived difficulty of the procedure was lower on average in the EG compared to the CG (3.52 vs. 4.39, 95% CI: 0.16-1.57, P=.016). There was no significant difference in terms of perceived difficulty using the equipment. The HCT measures reported by the EG group also presented a much lower dispersion, meaning a higher reliability, in determining the HCT value (3.10 vs. 26.94, SD; variances significantly different, P<.001, F: 75.25, Dfd: 68.19 for EG and CG). In the satisfaction test, the majority of the students in the EG reported that the experience was positive or very positive (80.7%) and reported that it had helped them to identify and use the equipment (78%) and to perform the exercise (66%). CONCLUSIONS: The simulation was well received by students in the EG, who felt more comfortable during the laboratory session, and it helped them to perform the exercise better, obtaining more accurate results, which indicates more effective training. EG students perceived the procedure as easier to perform, but did not report an improvement in the perceived difficulty in using the equipment. The increased reliability demonstrates that low-cost simulations are a good complement to the laboratory sessions.

Glycine-induced long-term synaptic potentiation is mediated by the glycine transporter GLYT1.

The negative symptoms of schizophrenia are reverted by treatment with glycine or other agonists of the glycine-B site which facilitate NMDA receptor function. On the other hand, there are experimental observations showing that exogenous application of glycine (0.5-10mM) results in a long-lasting potentiation of glutamatergic synaptic transmission (LTP-GLY). The characterization of the mechanisms underlying LTP-GLY could be useful to develop new therapies for schizophrenia. The main goal of this work is to deepen the understanding of this potentiation phenomenon. The present study demonstrates in rat hippocampal slices that superfusion of glycine 1mM during 30 min produces a potentiation of excitatory postsynaptic potentials in CA3-CA1 pathway lasting at least 1h. Glycine application does not modify neither presynaptic fiber volley nor paired-pulse facilitation of synaptic potentials. This LTP-GLY is independent of both strychnine-sensitive glycine receptors and nifedipine-sensitive calcium channels. Interestingly, LTP-GLY is not inhibited but strengthened by NMDA receptors antagonists such as AP-5 or MK-801. In contrast, LTP-GLY is partially or totally blocked with the antagonists of glycine transporter GLYT1, sarcosine or ALX-5407, respectively. These results indicate that LTP-GLY requires the activation of GLYT1, a glycine transporter co-localized and associated to NMDA receptors. In addition, the fact that NMDA receptor inhibition increases LTP-GLY magnitude, opens the possibility that these receptors could have a negative control on GLYT1 activity.

Role of taurine uptake on the induction of long-term synaptic potentiation.

Taurine application in the CA1 area of rat hippocampal slices induces a long-lasting potentiation of excitatory synaptic transmission that has some mechanistic similitude with the late phase of long-term potentiation (L-LTP). Previous indirect evidence such as temperature and sodium dependence indicated that taurine uptake is one of the primary steps leading to the taurine-induced synaptic potentiation. We show that taurine-induced potentiation is not related to the intracellular accumulation of taurine and is not impaired by 2-guanidinoethanesulphonic acid, a taurine transport inhibitor that is a substrate of taurine transporter. We have found that taurine uptake in hippocampal synaptosomes was inhibited by SKF 89976A, a GABA uptake blocker that is not transportable by GABA transporters. SKF 89976A prevents the induction of synaptic potentiation by taurine application. This effect is neither mimicked by nipecotic acid, a broad inhibitor of GABA transporters that does not affect taurine uptake, nor by NO-711, a specific and potent inhibitor of GABA transporter GAT-1. In addition, L-LTP induced by trains of high-frequency stimulation is also inhibited by SKF 89976A, and taurine, at a concentration that does not change basal synaptic transmission, overcomes such inhibition. We conclude that taurine induces synaptic potentiation through the activation of a system transporting taurine and that taurine uptake is required for the induction of synaptic plasticity phenomena such as L-LTP.