Computational identification of phospho-tyrosine sub-networks related to acanthocyte generation in neuroacanthocytosis

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.     

Analysis of the Role of Interleukin 6 Receptor Haplotypes in the Regulation of Circulating Levels of Inflammatory Biomarkers and Risk of Coronary Heart Disease

Variants at the interleukin 6 receptor (IL6R) gene regulate inflammation and are associated with risk of coronary heart disease (CHD). The aim of the present study was to investigate the effects of IL6R haplotypes on circulating levels of inflammatory biomarkers and risk of CHD. We performed a discovery analysis in SHEEP, a myocardial infarction (MI) case control study (n = 2,774) and replicated our results in two large, independent European populations, PROCARDIS, a CHD case control study (n = 7,998), and IMPROVE (n = 3,711) a prospective cardiovascular cohort study. Two major haplotype blocks (rs12083537A/G and rs4075015A/T—block 1; and rs8192282G/A, rs4553185T/C, rs8192284A/C, rs4240872T/C and rs7514452T/C—block 2) were identified in the IL6R gene. IL6R haplotype associations with C-reactive protein (CRP), fibrinogen, IL6, soluble IL6R (sIL6R), IL6, IL8 and TNF-α in SHEEP, CRP and fibrinogen in PROCARDIS and CRP in IMPROVE as well as association with risk of MI and CHD, were analyzed by THESIAS. Haplotypes in block 1 were associated neither with circulating inflammatory biomarkers nor with the MI/CHD risk. Haplotypes in block 2 were associated with circulating levels of CRP, in all three study populations, with fibrinogen in SHEEP and PROCARDIS, with IL8 and sIL6Rin SHEEP and with a modest, non significant, increase (7%) in MI/CHD risk in the three populations studied. Our results indicate that IL6R haplotypes regulate the circulating levels of inflammatory biomarkers. Lack of association with the risk of CHD may be explained by the combined effect of SNPs with opposite effect on the CHD risk, the sample size as well as by structural changes affecting sIL6R stability in the circulation.     

Detection of Autonomic Dysfunction in Hemodialysis Patients Using the Exercise Treadmill Test: The Role of the Chronotropic Index,Heart Rate Recovery,and R-R Variability

Autonomic dysfunction is highly prevalent in hemodialysis patients and has been implicated in their increased risk of cardiovascular mortality.

Objective

To evaluate the ability of different parameters of exercise treadmill test to detect autonomic dysfunction in hemodialysis patients.

Methods

Cross-sectional study involving hemodialysis patients and a control group. Clinical examination, blood sampling, echocardiogram, 24-hour Holter, and exercise treadmill test were performed. A ramp treadmill protocol symptom-limited with active recovery was employed.

Results

Forty-one hemodialysis patients and 41 controls concluded the study. There was significant difference between hemodialysis patients and controls in autonomic function parameters in 24h-Holter and exercise treadmill test. Probability of having autonomic dysfunction in hemodialysis patients compared to controls was 29.7 at the exercise treadmill test and 13.0 in the 24-hour Holter. Chronotropic index, heart rate recovery at the 1st min, and SDNN at exercise were used to develop an autonomic dysfunction score to grade autonomic dysfunction, in which, 83% of hemodialysis patients reached a scoring ≥2 in contrast to 20% of controls. Hemodialysis was independently associated with either altered chronotropic index or autonomic dysfunction scoring ≥2 in every tested model (OR=50.1, P=0.003; and OR=270.9, P=0.002, respectively, model 5).

Conclusion

The exercise treadmill test was feasible and useful to diagnose of the autonomic dysfunction in hemodialysis patients. Chronotropic index and autonomic dysfunction scoring ≥2 were the most effective parameters to differentiate between hemodialysis patients and controls suggesting that these variables portrays the best ability to detect autonomic dysfunction in this setting.     

Kidney Function,Endothelial Activation and Atherosclerosis in Black and White Africans with Rheumatoid Arthritis

Objective

To determine whether kidney function independently relates to endothelial activation and ultrasound determined carotid atherosclerosis in black and white Africans with rheumatoid arthritis (RA).

Methods

We calculated the Jelliffe, 5 Cockcroft-Gault equations, Salazar-Corcoran, Modification of Diet in Renal Disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) estimated glomerular filtration rate (EGFR) equations in 233 (112 black) RA patients.

Results

The CKD-EPI eGFR was <90 ml/min/1.73m² in 49.1% and 30.6% of black and white patients, respectively (odds ratio (95% confidence interval) = 2.19 (1.28–3.75), p = 0.004). EGFRs were overall consistently associated with monocyte chemoattractant protein-1 and angiopoietin 2 concentrations in white patients, and with carotid intima-media thickness and plaque in black participants. Amongst black patients, plaque prevalence was 36.7% and the area under the curve (AUC) of the receiver operating characteristic (ROC) curve was not associated with plaque presence for the MDRD equation (p = 0.3), whereas the respective relationship was significant or borderline significant (p = 0.003 to 0.08) and of similar extent (p>0.1 for comparisons of AUC (SE)) for the other 8 equations. Based on optimal eGFR cutoff values with sensitivities and specificities ranging from 42 to 60% and 70 to 91% respectively, as determined in ROC curve analysis, a low eGFR increased the odds ratio for plaque 2.2 to 4.0 fold.

Conclusion

Reduced kidney function is independently associated with atherosclerosis and endothelial activation in black and white Africans with RA, respectively. CKD is highly prevalent in black Africans with RA. Apart from the MDRD, eGFR equations are useful in predicting carotid plaque presence, a coronary heart disease equivalent, amongst black African RA patients.     

Toll-like receptor expression in normal ovary and ovarian tumors

Recent studies have implicated inflammation in the initiation and progression of ovarian cancer, though the mechanisms underlying this effect are still not clear. Toll-like receptors (TLRs) allow immune cells to recognize pathogens and to trigger inflammatory responses. Tumor cell expression of TLRs can promote inflammation and cell survival in the tumor microenvironment. Here we sought to characterize the expression of TLRs in normal human ovaries, benign and malignant ovarian tumors from patients, and in established ovarian tumor cell lines. We report that TLR2, TLR3, TLR4, and TLR5 are strongly expressed on the surface epithelium of normal ovaries. In contrast to previous studies of uterus and endocervix, we found no cyclic variation in TLR expression occurred in murine ovaries. TLR2, TLR3, TLR4, and TLR5 are expressed in benign conditions, epithelial tumors, and in ovarian cancer cell lines. Variable expression of TLR6 and TLR8 was seen in benign and malignant epithelium of some patients, while expression of TLR1, TLR7, and TLR9 was weak. Normal and malignant ovarian stroma were negative for TLR expression. Vascular endothelial cells, macrophages, and occasional fibroblasts in tumors were positive. Functional activity for TLRs was demonstrated by stimulation of cell lines with specific ligands and subsequent activation and translocation of NFκB and release of the proinflammatory cytokines interleukin-6 and CCL-2. These studies demonstrate expression of multiple TLRs in the epithelium of normal ovaries and in ovarian tumor cells, and may indicate a mechanism by which epithelial tumors manipulate inflammatory pathways to facilitate tumor progression.     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer

Introduction

Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO₂T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease.

Methods

We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO₂T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging.

Results

KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO₂T alone did not influence cancer progression, combining the KD with HBO₂T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls.

Conclusions

KD and HBO₂T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.     

Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.     

An Analysis of Marsupial Interordinal Relationships Based on 12S rRNA, tRNA Valine, 16S rRNA, and Cytochrome b Sequences

The basal split among living marsupials is traditionally placed between the cohorts Ameridelphiaand Australidelphia. Ameridelphia includes all American forms excepting the South AmericanDramicuipx gliroidex (Order Microbiotheria). Australidelphia includes all Australasian taxaplus Dromiciops glinmles. DNA data support Eometatheria Dromiciaps + Diprotodontia +Dasyuromorphia + Notoryctemorphia) but do not resolve the position of bandicoots, whetherwith other australidelphians or with ameridelphians. Also, the most robust molecular trees (DNAhybridization, multigene studies) exhibit minimal branch subdivision and raise the possibility ofartit'actual associations owing to long branch attraction. We analyzed data sets that consistedof complete sequences tor four niitochondrial genes (cytochrome b, 12S rRNA, tRNA valine,16S rRNA). One data set included 14 marsupial taxa. A second data set included 14 marsupialsas well as outgroup sequences (one monolreme; 20 placentals). Phylogenetic analyses includedparsimony, minimum evolution, maximum likelihood, and quartet puzzling. When phylogeneticanalyses were restricted to just the marsupial sequences, there was 75 to 96% boostrap supportfor the separation of Ameridelphia versus Australidelphia. This suggests that either one orboth of these groups are monophyletic. Also, there was 71 to 98% bootstrap support for theseparation of Eometatheria versus Ameridelphia + Peramelina. Nonmonophyly of several a prioriclades was accepted by at least some statistical tests including the following: Diprotodontia+ Peramelina, Notoryctemorphia + Peramelina, Diprotodonlia + Notoryctemorphia, and themonophyly of Australasian marsupials. With the inclusion of outgroup sequences, there wasreduced bootstrap support for associations among marsupial orders and statistical tests failed toreject all interordinal associations that were tested.