Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA₂s siRNA. cPLA₂ and iPLA₂ enzyme activities and cPLA₂ phosphorylation were stimulated after E. coli incubation and were attenuated by PLA₂, PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.     

Effectiveness of nursing interventions in heart failure patients in home care using NANDA-I,NIC, and NOC

ObjectiveThe objective of the study is to evaluate the effectiveness of nursing interventions (NIC) using nursing outcomes (NOC) and based on NANDA-I nursing diagnoses in patients with heart failure in home care.MethodIn this longitudinal study, 23 patients with heart failure were followed for 6 months, in four home visits. During the visits, nursing diagnoses were established, outcomes assessed, and interventions implemented.ResultsOf the 11 NIC interventions implemented, eight proved effective, that is, showed significant improvement between the first and the fourth visit, according to scores obtained for six outcomes: knowledge: treatment regimen, knowledge: medication, compliance behavior, symptom control, activity tolerance, and energy conservation.ConclusionNIC interventions health education, self-modification assistance, behavior modification, teaching: prescribed medication, teaching: disease process, nutritional counseling, telephone consultation, and energy conservation showed effective outcomes based on NOC scores, suggesting that the NANDA-I, NIC, and NOC linkage is useful in patients with heart failure in home care.     

What ethologically based models have taught us about the neural systems underlying fear and anxiety

Classical Pavlovian fear conditioning to painful stimuli has provided the generally accepted view of a core system centered in the central amygdala to organize fear responses. Ethologically based models using other sources of threat likely to be expected in a natural environment, such as predators or aggressive dominant conspecifics, have challenged this concept of a unitary core circuit for fear processing. We discuss here what the ethologically based models have told us about the neural systems organizing fear responses. We explored the concept that parallel paths process different classes of threats, and that these different paths influence distinct regions in the periaqueductal gray - a critical element for the organization of all kinds of fear responses. Despite this parallel processing of different kinds of threats, we have discussed an interesting emerging view that common cortical-hippocampal-amygdalar paths seem to be engaged in fear conditioning to painful stimuli, to predators and, perhaps, to aggressive dominant conspecifics as well. Overall, the aim of this review is to bring into focus a more global and comprehensive view of the systems organizing fear responses.     

Rotavirus serotypes and electropherotypes identified among hospitalised children in São Luís, Maranhão, Brazil

During June 1997-June 1999 rotavirus infection was screened in infants aged up to 2 years and hospitalised with acute diarrhoea in S?o Luís, Northeastern Brazil. Altogether, 128 stool samples were collected from diarrhoeic patients and additional 122 faecal specimens from age and temporal matched inpatients without diarrhoea were obtained; rotavirus positivity rates for these groups were 32.0% (41/128) and 9.8% (12/122), respectively (p < 0.001). Both electropherotyping and serotyping could be performed in 42 (79.2%) of the 53 rotavirus-positive stool samples. Long and short electropherotypes were detected at similar rates--38.1% and 40.5% of specimens, respectively. Overall, a G serotype could be assigned for 35 (83.3%) of specimens, the majority of them (66.7%) bearing G1-serotype specificity. Taking both electropherotypes and serotypes together, G1 rotavirus strains displaying long and short RNA patterns accounted for 30.9% and 19.0% of tested specimens, respectively; all G2 strains had short electropherotype. Rotavirus gastroenteritis was detected year-round and, in 1998, the incidence rates tended to be higher during the second semester than in the first semester: 45.2% and 26.1% (p = 0.13), respectively. Rotavirus infections peaked at the second semester of life with frequencies of 30.1% and 13.5% for diarrhoeic children and controls, respectively. While the six rotavirus strains bearing G2-type specificity were circulating throughout the whole study period, G1 serotypes (n = 27) emerged as from June 1998 onwards, 20 (74.1%) of which clustering in 1998. These data underscore the importance of rotaviruses in the aetiology of severe infantile gastroenteritis in Northeastern Brazil and sustain the concept that a future vaccine should confer protection against more than one serotype.     

Development of a Novel Human Cell-Derived Tissue-Engineered Heart Valve for Transcatheter Aortic Valve Replacement: an In Vitro and In Vivo Feasibility Study

Transcatheter aortic valve replacement (TAVR) is being extended to younger patients. However, TAVR-compatible bioprostheses are based on xenogeneic materials with limited durability. Off-the-shelf tissue-engineered heart valves (TEHVs) with remodeling capacity may overcome the shortcomings of current TAVR devices. Here, we develop for the first time a TEHV for TAVR, based on human cell-derived extracellular matrix and integrated into a state-of-the-art stent for TAVR. The TEHVs, characterized by a dense acellular collagenous matrix, demonstrated in vitro functionality under aortic pressure conditions (n?=?4). Next, transapical TAVR feasibility and in vivo TEHV functionality were assessed in acute studies (n?=?5) in sheep. The valves successfully coped with the aortic environment, showing normal leaflet motion, free coronary flow, and absence of stenosis or paravalvular leak. At explantation, TEHVs presented full structural integrity and initial cell infiltration. Its long-term performance proven, such TEHV could fulfill the need for next-generation lifelong TAVR prostheses.     

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/microL (equivalent to 2465.8 molecules/microL) to 10-9 (equivalent to 2.46 molecules/microL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/microL and for the nested PCR was 2.46 molecules/microL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.     

Homeostasis maintenance of encapsulated cells

Cell niche homeostasis plays a critical role in many bodily functions including tissue functionality, stem cell maintenance and differentiation, wound healing, cancer development and propagation, and many others. Many tissue engineering approaches overlook the importance of engineered constructs homeostasis, in particular for transplantation purposes. Here, we present a study of the effect of encapsulation duration on engineered tissue maturation and provide a protocol for the evaluation of critical conditions for transplantation purposes. In brief, SHSY5Y human neuroblastoma cells were encapsulated in 2% alginate by electrohydrodynamic jetting method for up to 4 weeks. We evaluated extracellular matrix niche formation and tissue maturation in situ through COL1A1 expression. In in vitro conditions, we studied the ability of cells to maintain their critical functions after being released from alginate beads. Cellular viability was evaluated via an apoptosis/necrosis detection kit and AlamarBlue assay, and functionality via immunocytochemistry. We proved the importance of engineered tissue homeostasis stabilization for future cell recovery, in particular, for our system cells encapsulated for 28 days met all critical requirements for successful tissue transplantation. Maturation of engineered tissue constructs could be accelerated by enriching alginate with growth factors or extracellular matrix molecules.     

A mixed‐effect model for positive responses augmented by zeros

In this research article, we propose a class of models for positive and zero responses by means of a zero‐augmented mixed regression model. Under this class, we are particularly interested in studying positive responses whose distribution accommodates skewness. At the same time, responses can be zero, and therefore, we justify the use of a zero‐augmented mixture model. We model the mean of the positive response in a logarithmic scale and the mixture probability in a logit scale, both as a function of fixed and random effects. Moreover, the random effects link the two random components through their joint distribution and incorporate within‐subject correlation because of the repeated measurements and between‐subject heterogeneity. A Markov chain Monte Carlo algorithm is tailored to obtain Bayesian posterior distributions of the unknown quantities of interest, and Bayesian case‐deletion influence diagnostics based on the q‐divergence measure is performed. We apply the proposed method to a dataset from a 24hour dietary recall study conducted in the city of São Paulo and present a simulation study to evaluate the performance of the proposed methods. Copyright © 2015 John Wiley & Sons, Ltd.