Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB

Confiabilidad de un instrumento para clasificar al recién nacido de acuerdo con la complejidad de la atención

Na maioria das maternidades, a classificação, a avaliação e a definição da unidade a encaminhar o recém-nascido (RN) após o nascimento são realizadas pelo médico. A\ud avaliação ocorre na sala de parto considerando: peso ao nascer; idade gestacional;\ud condutas que definem quadro clínico e doença. Este estudo observacional teve\ud como objetivo avaliar a confiabilidade de um instrumento de classificação de RN. A pesquisa foi realizada no berçário de um hospital público, São Paulo. Nove enfermeiras\ud aplicaram o instrumento a 63 RN, sendo duas simultaneamente em cada um dos\ud cinco setores do berçário. Verificou-se que o nível de concordância Kappa entre as enfermeiras foi excelente para a maioria das áreas de cuidado (69,0%). Concluiu-se que houve consenso e concordância das enfermeiras quanto ao instrumento ser completo, de fácil entendimento e aplicável, porém despende muito tempo. As enfermeiras reconhecem a importância do instrumento para o dimensionamento dos profissionais, organização e planejamento do cuidado.In most maternity units, the physician classifies, evaluates, and determines which unit will receive the newborn (NB) after birth. Evaluation occurs in the delivery room, taking into consideration the following factors: birth weight, gestational age, and behaviors that define the clinical picture and disease. This observational study evaluates the reliability of an NB classification instrument. The study was conducted at the nursery of a public hospital in São Paulo. Nine nurses applied the instrument to 63 NB, with two of the nurses working simultaneously in each of the nursery’s fi ve sectors. The Kappa level of agreement among the nurses was found to be excellent for most care areas\ud (69.0%). It was concluded that there was a consensus and agreement among the\ud nurses that the instrument was complete, easy to understand and applicable, but was\ud very time consuming. The nurses recognize the instrument’s importance for the allocation of professionals, organization, and care planning.En la mayoría de las maternidades la clasificación, la evaluación y la definición de la unidad para referir el recién nacido (RN), son realizadas por el médico. La evaluación se realiza en la sala de parto, considerando: peso al nacer, edad gestacional y conductas que definen el cuadro clínico y la enfermedad. Este estudio observacional tuvo como objetivo evaluar la confiabilidad de un instrumento de clasificación del RN. Fue realizada en el servicio de neonatología de un hospital público en Sao Paulo. Nueve enfermeras\ud aplicaron el instrumento a 63 RN, siendo aplicados dos de forma simultánea en los cinco sectores de la unidad neonatal. El nivel de concordancia Kappa fue excelente para la mayoría de las áreas de atención (69,0%). Se concluyó que hubo consenso y\ud concordancia entre las enfermeras, quienes expresaron que el instrumento es completo, fácil de entender y de aplicar, pero se necesita mucho tempo. Las enfermeras reconocen la importancia de este instrumento para dimensionar el número de profesionales, la organización y la planificación de la atención

Early epilepsy in children with Zika-related microcephaly in a cohort in Recife, Brazil: Characteristics, electroencephalographic findings, and treatment response.

OBJECTIVE: To estimate the incidence of epilepsy in children with Zika-related microcephaly in the first 24 months of life; to characterize the associated clinical and electrographic findings; and to summarize the treatment responses. METHODS: We followed a cohort of children, born during the 2015-2016 Zika virus (ZIKV) epidemic in Brazil, with congenital microcephaly and evidence of congenital ZIKV infection on neuroimaging and/or laboratory testing. Neurological assessments were performed at ≤3, 6, 12, 15, 18, 21, and 24 months of life. Serial electroencephalograms were performed over the first 24 months. RESULTS: We evaluated 91 children, of whom 48 were female. In this study sample, the cumulative incidence of epilepsy was 71.4% in the first 24 months, and the main type of seizure was infantile spasms (83.1%). The highest incidence of seizures occurred between 3 and 9 months of age, and the risk remained high until 15 months of age. The incidence of infantile spasms peaked between 4 and 7 months and was followed by an increased incidence of focal epilepsy cases after 12 months of age. Neuroimaging results were available for all children, and 100% were abnormal. Cortical abnormalities were identified in 78.4% of the 74 children evaluated by computed tomography and 100% of the 53 children evaluated by magnetic resonance imaging. Overall, only 46.1% of the 65 children with epilepsy responded to treatment. The most commonly used medication was sodium valproate with or without benzodiazepines, levetiracetam, phenobarbital, and vigabatrin. SIGNIFICANCE: Zika-related microcephaly was associated with high risk of early epilepsy. Seizures typically began after the third month of life, usually as infantile spasms, with atypical electroencephalographic abnormalities. The seizure control rate was low. The onset of seizures in the second year was less frequent and, when it occurred, presented as focal epilepsy

Heat transfer in solar absorber plates with micro-channels

Analytical, computational and experimental studies were carried out to investigate heat transfer and fluid flow in micro-channel absorber plates for compact (thin and light-weight) solar thermal collectors. The main objective of the work was to study different design and/or operating scenarios as well as study the significance of various micro-scaling effects. Analytical investigation showed that, under similar conditions, the proposed design yields a much higher fin efficiency, F and collector efficiency factor, F’ compared with the conventional solar collector design. An analytical model combining convective heat transfer in the collector fluid with axial conduction in the metal plate was developed. The predicted plate temperature profiles from the analytical model were in close agreement with the measured profiles. The model further showed that axial thermal conduction can significantly alter the plate temperature profile. Experiments were designed to represent real life operation of the proposed system. A CFD study, using the same design and operating parameters, produced results comparable with experiments. This numerical simulation also gave further insight into the heat transfer and fluid flow patterns in the micro-channel plate. The effect of channel cross section geometry was studied. The Nusselt number was observed to increase as the aspect ratio approached unity. Measured friction factors were similar in trend to the predictions for rectangular channels, although the overall rise in fluid temperature resulted in slightly lower friction factors. Thermal performance reduced slightly with increase in hydraulic diameter. The significance of various scaling effects was also investigated experimentally and numerically. Most of the typical scaling effects such as viscous dissipation and entrance effects were found to be insignificant however, conjugate heat transfer, surface boundary condition, surface finish and measurement uncertainties could be significant. The results showed a Reynolds number dependent Nusselt number which has been attributed to axial thermal conduction. It was also observed that only three walls were transferring heat; the walls of heat transfer had a uniform peripheral temperature while the heat flux varied peripherally. The closest simplified thermal boundary condition to represent heat transfer in these channels is the H1 with three (3) walls transferring heat. Increased surface roughness (obtained by using an etching technique to create the channels) was found to have a detrimental effect on heat transfer. The results showed that thermal improvement can be achieved by increasing the fluid velocity; however, pumping the thermal fluid above a pump power per plate area of 0.3 W/m2 resulted in marginal improvement. In practice, optimum microchannel geometry in plates should be sized based on fluid properties and operating conditions. The micro-channels should also have thin walls to minimise the effects of conjugate heat transfer. A Photovoltaic pump should be installed alongside the collector in order to provide pumping power required and minimise the overall fluid temperature rise. The results are beneficial for the design of micro-channel absorber plates for low heat flux operation up to 1000W/m2

Sodium bicarbonate supplementation improves severe-intensity intermittent exercise under moderate acute hypoxic conditions

Acute moderate hypoxic exposure can substantially impair exercise performance, which occurs with a concurrent exacerbated rise in hydrogen cation (H+) production. The purpose of this study was therefore, to alleviate this acidic stress through sodium bicarbonate (NaHCO3) supplementation and determine the corresponding effects on severe intensity intermittent exercise performance. Eleven recreationally active individuals participated in this randomised, double-blind, crossover study performed under acute normobaric hypoxic conditions (FiO2% = 14.5%). Pre-experimental trials involved the determination of time to attain peak bicarbonate anion concentrations ([HCO3-]) following NaHCO3 ingestion. The intermittent exercise tests involved repeated 60 s work in their severe intensity domain and 30 s recovery at 20 W to exhaustion. Participants ingested either 0.3 g·kg bm-1 of NaHCO3 or a matched placebo of 0.21 g·kg bm-1 of sodium chloride prior to exercise. Exercise tolerance (+110.9 ± 100.6 s; 95% CI: 43.3 to 178 s; g = 1.0) and work performed in the severe intensity domain (+5.8 ± 6.6 kJ; 95% CI: 1.3 to 9.9 kJ; g = 0.8) were enhanced with NaHCO3 supplementation. Furthermore, a larger post-exercise blood lactate concentration was reported in the experimental group (+4 ± 2.4 mmol·l-1; 95% CI: 2.2 to 5.9; g = 1.8), while blood [HCO3-] and pH remained elevated in the NaHCO3 condition throughout experimentation. In conclusion, this study reported a positive effect of NaHCO3 under acute moderate hypoxic conditions during intermittent exercise and therefore, may offer an ergogenic strategy to mitigate hypoxic induced declines in exercise performance

Quantitative Proteomic and Interaction Network Analysis of Cisplatin Resistance in HeLa Cells

Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts

Maternal Obesity during Gestation Impairs Fatty Acid Oxidation and Mitochondrial SIRT3 Expression in Rat Offspring at Weaning

In utero exposure to maternal obesity increases the offspring's risk of obesity in later life. We have also previously reported that offspring of obese rat dams develop hepatic steatosis, mild hyperinsulinemia, and a lipogenic gene signature in the liver at postnatal day (PND)21. In the current study, we examined systemic and hepatic adaptations in male Sprague-Dawley offspring from lean and obese dams at PND21. Indirect calorimetry revealed decreases in energy expenditure (p<0.001) and increases in RER values (p<0.001), which were further exacerbated by high fat diet (45% kcals from fat) consumption indicating an impaired ability to utilize fatty acids in offspring of obese dams as analyzed by PRCF. Mitochondrial function is known to be associated with fatty acid oxidation (FAO) in the liver. Several markers of hepatic mitochondrial function were reduced in offspring of obese dams. These included SIRT3 mRNA (p = 0.012) and mitochondrial protein content (p = 0.002), electron transport chain complexes (II, III, and ATPase), and fasting PGC-1α mRNA expression (p<0.001). Moreover, hepatic LCAD, a SIRT3 target, was not only reduced 2-fold (p<0.001) but was also hyperacetylated in offspring of obese dams (p<0.005) suggesting decreased hepatic FAO. In conclusion, exposure to maternal obesity contributes to early perturbations in whole body and liver energy metabolism. Mitochondrial dysfunction may be an underlying event that reduces hepatic fatty acid oxidation and precedes the development of detrimental obesity associated co-morbidities such as insulin resistance and NAFLD