Randomized controlled trial assessing the effect of simvastatin in primary biliary cirrhosis

Background This study evaluated the effect of statins in Primary biliary cirrhosis (PBC) on endothelial function, anti-oxidant status and vascular compliance. Methods Primary biliary cirrhosis patients with hypercholesterolaemia were randomized to receive 20mg simvastatin or placebo in a single blind, randomized controlled trial. Body mass index, blood pressure, glucose, liver function, lipid profile, immunoglobulin levels, serological markers of endothelial function and anti-oxidant status were measured as well as vascular compliance, calculated from pulse wave analysis and velocity, at recruitment and again at 3, 6, 9 and 12months. Results Twenty-one PBC patients (F=20, mean age = 55) were randomized to simvastatin 20mg (n=11) or matched placebo (n=10). At completion of the trial, serum cholesterol levels in the simvastatin group were significantly lower compared with the placebo group (4.91mmol/L vs. 6.15mmol/L, P=0.01). Low-density lipoprotein (LDL) levels after 12months were also significantly lower in the simvastatin group (2.33mmol/L vs. 3.53mmol/L, P=0.01). After 12months of treatment, lipid hydroperoxides were lower (0.49mol/L vs. 0.59mol/L, P=0.10) while vitamin C levels were higher (80.54mol/L vs. 77.40mol/L, P=0.95) in the simvastatin group. Pulse wave velocity remained similar between treatment groups at 12months (8.45m/s vs. 8.80m/s, P=0.66). Only one patient discontinued medication owing to side effects. No deterioration in liver transaminases was noted in the simvastatin group. Conclusions Statin therapy in patients with PBC appears safe and effective towards overall reductions in total cholesterol and LDL levels. Our initial study suggests that simvastatin may also confer advantageous effects on endothelial function and antioxidant status.

Ultrasound entropy may be a new non-invasive measure of pre-clinical vascular damage in young hypertensive patients

Background: The identification of pre-clinical microvascular damage in hypertension by non-invasive techniques has proved frustrating for clinicians. This proof of concept study investigated whether entropy, a novel summary measure for characterizing blood velocity waveforms, is altered in participants with hypertension and may therefore be useful in risk stratification.

Methods: Doppler ultrasound waveforms were obtained from the carotid and retrobulbar circulation in 42 participants with uncomplicated grade 1 hypertension (mean systolic/diastolic blood pressure (BP) 142/92 mmHg), and 26 healthy controls (mean systolic/diastolic BP 116/69 mmHg). Mean wavelet entropy was derived from flow-velocity data and compared with traditional haemodynamic measures of microvascular function, namely the resistive and pulsatility indices.

Results: Entropy, was significantly higher in control participants in the central retinal artery (CRA) (differential mean 0.11 (standard error 0.05 cms(-1)), CI 0.009 to 0.219, p 0.017) and ophthalmic artery (0.12 (0.05), CI 0.004 to 0.215, p 0.04). In comparison, the resistive index (0.12 (0.05), CI 0.005 to 0.226, p 0.029) and pulsatility index (0.96 (0.38), CI 0.19 to 1.72, p 0.015) showed significant differences between groups in the CRA alone. Regression analysis indicated that entropy was significantly influenced by age and systolic blood pressure (r values 0.4-0.6). None of the measures were significantly altered in the larger conduit vessel.

Conclusion: This is the first application of entropy to human blood velocity waveform analysis and shows that this new technique has the ability to discriminate health from early hypertensive disease, thereby promoting the early identification of cardiovascular disease in a young hypertensive population.

O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

Physical rehabilitation interventions for adult intensive care patients across the continuum of recovery (i.e. within the intensive care unit (ICU) and following ICU and hospital discharge): an overview of systematic reviews

Background
Patients admitted to the intensive care unit with critical illness often experience significant physical impairments, which typically persist for many years following resolution of the original illness. Physical rehabilitation interventions that enhance restoration of physical function have been evaluated across the continuum of recovery following critical illness including within the intensive care unit, following discharge to the ward and beyond hospital discharge. Multiple systematic reviews have been published appraising the expanding evidence investigating these physical rehabilitation interventions, although there appears to be variability in review methodology and quality. We aim to conduct an overview of existing systematic reviews of physical rehabilitation interventions for adult intensive care patients across the continuum of recovery.

Methods/design
This protocol has been developed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol (PRISMA-P) guidelines. We will search the Cochrane Systematic Review Database, Database of Abstracts of Reviews of Effectiveness, Cochrane Central Register of Controlled Trials, MEDLINE, Excerpta Medica Database and Cumulative Index to Nursing and Allied Health Literature databases. We will include systematic reviews of randomised controlled trials of adult patients, admitted to the intensive care unit and who have received physical rehabilitation interventions at any time point during their recovery. Data extraction will include systematic review aims and rationale, study types, populations, interventions, comparators, outcomes and quality appraisal method. Primary outcomes of interest will focus on findings reflecting recovery of physical function. Quality of reporting and methodological quality will be appraised using the PRISMA checklist and the Assessment of Multiple Systematic Reviews tool.

Discussion
We anticipate the findings from this novel overview of systematic reviews will contribute to the synthesis and interpretation of existing evidence regarding physical rehabilitation interventions and physical recovery in post-critical illness patients across the continuum of recovery.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Wavelet entropy of Doppler ultrasound blood velocity flow waveforms distinguishes nitric oxide-modulated states

Wavelet entropy assesses the degree of order or disorder in signals and presents this complex information in a simple metric. Relative wavelet entropy assesses the similarity between the spectral distributions of two signals, again in a simple metric. Wavelet entropy is therefore potentially a very attractive tool for waveform analysis. The ability of this method to track the effects of pharmacologic modulation of vascular function on Doppler blood velocity waveforms was assessed. Waveforms were captured from ophthalmic arteries of 10 healthy subjects at baseline, after the administration of glyceryl trinitrate (GTN) and after two doses of N(G)-nitro-L-arginine-methyl ester (L-NAME) to produce vasodilation and vasoconstriction, respectively. Wavelet entropy had a tendency to decrease from baseline in response to GTN, but significantly increased after the administration of L-NAME (mean: 1.60 ± 0.07 after 0.25 mg/kg and 1.72 ± 0.13 after 0.5 mg/kg vs. 1.50 ± 0.10 at baseline, p < 0.05). Relative wavelet entropy had a spectral distribution from increasing doses of L-NAME comparable to baseline, 0.07 ± 0.04 and 0.08 ± 0.03, respectively, whereas GTN had the most dissimilar spectral distribution compared with baseline (0.17 ± 0.08, p = 0.002). Wavelet entropy can detect subtle changes in Doppler blood velocity waveform structure in response to nitric-oxide-mediated changes in arteriolar smooth muscle tone.

Endothelial Function in Hypertension: Victim or Culprit?

Far from simply lining the inner surface of blood vessels, the cellular monolayer that comprises the endothelium is a highly active organ that regulates vascular tone. In health, the endothelium maintains the balance between opposing dilator and constrictor influences, while in disease, it is the common ground on which cardiovascular risk factors act to initiate the atherosclerotic process. As such, it is the site at which cardiovascular disease begins and consequently acts as a barometer of an individual's likely future cardiovascular health. The vascular endothelium is a very active organ responsible for the regulation of vascular tone through the effects of locally synthesized mediators, predominantly nitric oxide (NO), endothelial NO synthase (eNOS), and superoxide. NO is abundantly evident in normally functioning vasculature where it acts as a vasodilator, inhibits inflammation, and has an antiaggregant effect on platelets. Its depletion is both a sign and cause of endothelial dysfunction resulting from reduced activity of eNOS and amplified production of nicotinamide adenine dinucleotide oxidase, which, in turn, results in raised levels of reactive oxygen species. This cascade is the basis for reduced vascular compliance through an imbalanced regulation of tone with a predominance of vasoconstrictive elements. Further, structural changes in the microvasculature are a critical early step in the loss of normal function. This microvascular dysfunction is known to be highly predictive of future macrovascular events and is consequently a very attractive target for intervention in the hypertensive population in order to prevent cardiovascular events.

Optimising the management of polycystic kidney disease

Polycystic kidney disease (PKD) is the most common inherited renal disorder that results in chronic kidney disease. Clinical features include visible haematuria, loin pain, UTI and hypertension. The typical clinical course is a progressive increase in the number and size of renal cysts associated with gradual loss of kidney function (falling eGFR).

Differential requirements of aPKC activity within different epithelial tissues

Epithelial tissues are essential during morphogenesis and organogenesis. During development, epithelial tissues undergo several different remodeling processes, from cell intercalation to cell change shape. An epithelial cell has a highly polarized structure, which is important to maintain tissue integrity. The mechanisms that regulate and maintain apicobasal polarity and epithelial integrity are mostly conserved among all species and in different tissues within the same organism. aPKC-PAR complex localizes in the apical domain of polarized cells, and its function is essential for apicobasal polarization and epithelial integrity. In this work we characterized two novel alleles of aPKC: a temperature sensitive allele (aPKCTS), which has a point mutation on a kinase domain, and another allele with a point mutation on a highly conserved amino acid within the PB1 domain of aPKC (aPKCPB1). Analysis of the aPKCTS mutant phenotypes, lead us to propose that during development different epithelial tissues have differential requirements of aPKC activity. More specifically, our work suggests de novo formation of adherens junctions (AJs) is particularly sensitive to sub-optimal levels of apkc activity. Analysis of the aPKCPB1 allele, suggests that aPKC is likely to have an apical structural function mostly independent of its kinase activity. Altogether our work suggests that although loss of aPKC function is associated to similar epithelial phenotypes (e.g., loss of apicobasal polarization and epithelial integrity), the requirements of aPKC activity within these tissues are nevertheless likely to vary.

Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons

Among the various proteins which are induced when human cells are are treatened with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological functionand its possible contribution to the various antiviral and non-antiviral actions exerted by interferons.

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Dual studies on autonomic function and cerebral autoregulation in ischemic stroke patients

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2015

Grid structure impact in sparse point representation of derivatives

In the Sparse Point Representation (SPR) method the principle is to retain the function data indicated by significant interpolatory wavelet coefficients, which are defined as interpolation errors by means of an interpolating subdivision scheme. Typically, a SPR grid is coarse in smooth regions, and refined close to irregularities. Furthermore, the computation of partial derivatives of a function from the information of its SPR content is performed in two steps. The first one is a refinement procedure to extend the SPR by the inclusion of new interpolated point values in a security zone. Then, for points in the refined grid, such derivatives are approximated by uniform finite differences, using a step size proportional to each point local scale. If required neighboring stencils are not present in the grid, the corresponding missing point values are approximated from coarser scales using the interpolating subdivision scheme. Using the cubic interpolation subdivision scheme, we demonstrate that such adaptive finite differences can be formulated in terms of a collocation scheme based on the wavelet expansion associated to the SPR. For this purpose, we prove some results concerning the local behavior of such wavelet reconstruction operators, which stand for SPR grids having appropriate structures. This statement implies that the adaptive finite difference scheme and the one using the step size of the finest level produce the same result at SPR grid points. Consequently, in addition to the refinement strategy, our analysis indicates that some care must be taken concerning the grid structure, in order to keep the truncation error under a certain accuracy limit. Illustrating results are presented for 2D Maxwell's equation numerical solutions.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.