An image processing application for quantification of protein aggregates in Caenorhabditis elegans

Protein aggregation became a widely accepted marker of many polyQ disorders, including Machado-Joseph disease (MJD), and is often used as readout for disease progression and development of therapeutic strategies. The lack of good platforms to rapidly quantify protein aggregates in a wide range of disease animal models prompted us to generate a novel image processing application that automatically identifies and quantifies the aggregates in a standardized and operator-independent manner. We propose here a novel image processing tool to quantify the protein aggregates in a Caenorhabditis elegans (C. elegans) model of MJD. Confocal mi-croscopy images were obtained from animals of different genetic conditions. The image processing application was developed using MeVisLab as a platform to pro-cess, analyse and visualize the images obtained from those animals. All segmenta-tion algorithms were based on intensity pixel levels.The quantification of area or numbers of aggregates per total body area, as well as the number of aggregates per animal were shown to be reliable and reproducible measures of protein aggrega-tion in C. elegans. The results obtained were consistent with the levels of aggrega-tion observed in the images. In conclusion, this novel imaging processing applica-tion allows the non-biased, reliable and high throughput quantification of protein aggregates in a C. elegans model of MJD, which may contribute to a significant improvement on the prognosis of treatment effectiveness for this group of disor-ders

Automatic segmentation and 3D feature extraction of protein aggregates in Caenorhabditis Elegans

In the last years, it has become increasingly clear that neurodegenerative diseases involve protein aggregation, a process often used as disease progression readout and to develop therapeutic strategies. This work presents an image processing tool to automatic segment, classify and quantify these aggregates and the whole 3D body of the nematode Caenorhabditis Elegans. A total of 150 data set images, containing different slices, were captured with a confocal microscope from animals of distinct genetic conditions. Because of the animals’ transparency, most of the slices pixels appeared dark, hampering their body volume direct reconstruction. Therefore, for each data set, all slices were stacked in one single 2D image in order to determine a volume approximation. The gradient of this image was input to an anisotropic diffusion algorithm that uses the Tukey’s biweight as edge-stopping function. The image histogram median of this outcome was used to dynamically determine a thresholding level, which allows the determination of a smoothed exterior contour of the worm and the medial axis of the worm body from thinning its skeleton. Based on this exterior contour diameter and the medial animal axis, random 3D points were then calculated to produce a volume mesh approximation. The protein aggregations were subsequently segmented based on an iso-value and blended with the resulting volume mesh. The results obtained were consistent with qualitative observations in literature, allowing non-biased, reliable and high throughput protein aggregates quantification. This may lead to a significant improvement on neurodegenerative diseases treatment planning and interventions prevention

Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5–10.8% of fat) followed by fins (3.4–6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4–4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n -6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n 6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish – the muscle.

Nutritional composition of low protein and phenylalanine-restricted dishes prepared for phenylketonuric patients

Nutritional management is essential for Phenylketonuria (PKU) treatment, consisting in a semi-synthetic and low phenylalanine (Phe) diet, which includes strictly controlled amounts of low protein natural foods (essentially fruits and vegetables) supplemented with Phe-free protein substitutes and dietetic low-protein products. PKU diet has to be carefully planned, providing the best ingredient combinations, so that patients can achieve good metabolic control and an adequate nutritional status. Hereupon, it is mandatory to know the detailed composition of natural and/or cooked foodstuffs prepared specifically for these patients. We intended to evaluate sixteen dishes specifically prepared for PKU patients, regarding the nutritional composition, Phe and tyrosine (Tyr) contents, fatty acids profile, and vitamins E and B12 amounts. The nutritional composition of the cooked samples was 15.5–92.0 g/100 g, for moisture; 0.7–3.2 g/100 g, for protein; 0.1–25.0 g/100 g, for total fat; and 5.0–62.0 g/100 g, for total carbohydrates. Fatty acids profile and vitamin E amount reflected the type of fat used. All samples were poor in vitamin B12 (0.3–0.8 μg/100 g). Boiled rice presented the highest Phe content: 50.3 mg/g of protein. These data allow a more accurate calculation of the diet portions to be ingested by the patients according to their individual tolerance.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

C-reactive protein at 24 hours after hospital admission may have relevant prognostic accuracy in acute pancreatitis: a retrospective cohort study

Introduction: C-reactive protein (CRP) and Bedside Index for Severity in Acute Pancreatitis (BISAP) have been used in early risk assessment of patients with AP. Objectives: We evaluated prognostic accuracy of CRP at 24 hours after hospital admission (CRP24) for in-hospital mortality (IM) in AP individually and with BISAP. Materials and Methods: This retrospective cohort study included 134 patients with AP from a Portuguese hospital in 2009---2010. Prognostic accuracy assessment used area under receiver---operating characteristic curve (AUC), continuous net reclassification improvement (NRI), and integrated discrimination improvement (IDI). Results: Thirteen percent of patients had severe AP, 26% developed pancreatic necrosis, and 7% died during index hospital stay. AUCs for CRP24 and BISAP individually were 0.80 (95% confidence interval (CI) 0.65---0.95) and 0.77 (95% CI 0.59---0.95), respectively. No patients with CRP24 <60 mg/l died (P = 0.027; negative predictive value 100% (95% CI 92.3---100%)). AUC for BISAP plus CRP24 was 0.81 (95% CI 0.65---0.97). Change in NRI nonevents (42.4%; 95% CI, 24.9---59.9%) resulted in positive overall NRI (31.3%; 95% CI, − 36.4% to 98.9%), but IDI nonevents was negligible (0.004; 95% CI, − 0.007 to 0.014). Conclusions: CRP24 revealed good prognostic accuracy for IM in AP; its main role may be the selection of lowest risk patients.

Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Serum proteins and fractions, HDL-cholesterol and total IgG and IgE levels in cases of acute and chronic paracoccidioidomycosis

This study evaluated serum protein fractions, HDL-cholesterol, total immunoglobulin G and total immunoglobulin E levels in patients with acute and chronic paracoccidioidomycosis, by means of electrophoresis, enzymatic reaction and immunoenzymatic assay. The results demonstrated elevated levels of total immunoglobulin G, total immunoglobulin E, alpha-2 and gamma-globulins, which were more evident in acute than in chronic PCM, but no increase in HDL-cholesterol levels. There was a correlation between the levels of total immunoglobulin E and gamma-globulins and the alpha-2 and beta-globulin fractions in the acute form and between beta and gamma-globulins in both the acute and the chronic form. In conclusion, changes in total immunoglobulin G and immunoglobulin E levels and in the electrophoretic profile may be important markers for the prognosis and therapeutic follow-up of PCM cases, especially because protein electrophoresis is a simple laboratory test that can be applied when specific PCM serological tests are not available. In addition, levels of the gamma-globulin fraction greater than 2.0g/dl may suggest that the patient is developing a more severe form of PCM.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

Generation and phenotypic analysis of protein S-deficient mice.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

No role for CSF myelin basic protein levels in patients treated for childhood acute lymphoblastic leukemia

RESUME Introduction : La prophylaxie du système nerveux central lors d'un diagnostic de leucémie lymphoblastique aiguë de l'enfant a permis de réduire le risque de rechute mais a été associée dans certains cas à des neurotoxicités cliniques ou des anomalies radiologiques. Des moyens de prédire ces neurotoxicités font défaut, en particulier en raison de l'absence de corrélation claire entre les signes cliniques et les images radiologiques. Quelques auteurs ont suggéré que les taux de protéine basique de la myéline (MBP) mesurés dans le liquide céphalo-rachidien pouvaient avoir un intérêt dans ce contexte. Uné étude rétrospective de ces taux en relation avec des données cliniques et radiologiques est présentée dans ce travail. Matériel et Méthodes : Les taux de MBP mesurés dans le liquide céphalo-rachidien lors d'administration de chimiothérapie intrathécale, les examens cliniques neurologiques et les rapports radiologiques ont été rétrospectivement étudiés chez nos patients. Les données concernant des difficultés académiques éventuelles, ainsi que le niveau académique atteint ont été récoltées par l'intermédiaire de contacts téléphoniques réguliers organisés dans le cadre du suivi à long terme de nos patients. Résultats : Un total de 1248 dosages de MBP chez 83 patients, 381 examens neurologiques chez 34 patients et 69 rapports d'investigations neuroradiologiques chez 27 patients ont été analysés. Cinquante-deux patients ont eut au moins un taux anormal de MBP. Des anomalies radiologiques ont été décrites chez 47% de ces patients, parmi lesquels 14% ont présenté des difficultés scolaires sous une forme ou sous une autre. La proportion de patients ayant présenté des difficultés scolaires dans les groupes avec taux de MBP normal mais sans anomalies radiologiques décrites ou sans investigations radiologiques étaient respectivement de 0% et 3%, inférieurs dans tous les cas au groupe avec des taux normaux de MBP (100%, 22% and 5% respectivement). Discussion : Tout en prenant en compte les limitations dues à l'aspect rétrospectif de cette étude, nous avons conclu à une utilité limitée de ces dosages systématiques comme indicateur d'une neurotoxicité induite parle traitement dans le contexte de nos patients oncologiques. ABSTRACT Introduction : Central nervous system (CSF) prophylaxis of childhood acute lymphoblastic leukemia has dropped rates of relapses but has been associated wíth neurotoxicity and imaging abnormalities. Predictors of neurotoxícity are lacking, because of inconsistency between clinical symptoms and imaging. Some have suggested CSF Myelin Basic Protein (MBP) levels to be of potential interest. A retrospective analysis of MBP levels in correlation with clinical and radiological data is presented. Materials and Methods : MBP levels obtained at the time of intrathecals, charts, and neuroradiology reports were retrospectively analyzed. Academic achievement data were obtained from phone contacts with patients and families. Results : We retrieved 1248 dosages of MBP in 83 patients, 381 neurological exams in 34 patients and 69 neuroradiological investigations in 27 patients. Fifty-two patients had abnormal MBP levels. Radiological anomalies were present in 47% of those investigated, 14% of them having school difficulties. Proportions of patients with school difficulties in the groups with abnormal MBP levels but no radiological anomalies or with no radiological investigations were 0% and 3% respectively, which was lower than in the group of patients with normal MBP levels (100%, 22% and 5% respectively). Discussion : Notwithstanding the retrospective character of our study, we conclude that there is limited usefulness of systematic dosage of MBP as indicator of treatment-induced neurotoxicity in ALL patients.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

Use of a renal-specific oral supplement by haemodialysis patients with low protein intake does not increase the need for phosphate binders and may prevent a decline in nutritional status and quality of life.

BACKGROUND: Protein-energy wasting is a frequent and debilitating condition in maintenance dialysis. We randomly tested if an energy-dense, phosphate-restricted, renal-specific oral supplement could maintain adequate nutritional intake and prevent malnutrition in maintenance haemodialysis patients with insufficient intake. METHODS: Eighty-six patients were assigned to a standard care (CTRL) group or were prescribed two 125-ml packs of Renilon 7.5(R) daily for 3 months (SUPP). Dietary intake, serum (S) albumin, prealbumin, protein nitrogen appearance (nPNA), C-reactive protein, subjective global assessment (SGA) and quality of life (QOL) were recorded at baseline and after 3 months. RESULTS: While intention to treat analysis (ITT) did not reveal strong statistically significant changes in dietary intake between groups, per protocol (PP) analysis showed that the SUPP group increased protein (P < 0.01) and energy (P < 0.01) intakes. In contrast, protein and energy intakes further deteriorated in the CTRL group (PP). Although there was no difference in serum albumin and prealbumin changes between groups, in the total population serum albumin and prealbumin changes were positively associated with the increment in protein intake (r = 0.29, P = 0.01 and r = 0.27, P = 0.02, respectively). The SUPP group did not increase phosphate intake, phosphataemia remained unaffected, and the use of phosphate binders remained stable or decreased. The SUPP group exhibited improved SGA and QOL (P < 0.05). CONCLUSION: This study shows that providing maintenance haemodialysis patients with insufficient intake with a renal-specific oral supplement may prevent deterioration in nutritional indices and QOL without increasing the need for phosphate binders.

Influence of inflammation on total energy expenditure in hemodialysis patients.

Background and Objectives: Studies show that inflammation can contribute to an increase in resting energy expenditure in patients with chronic kidney disease; however, findings about total energy expenditure (TEE) have not been reported. The aim of this study was to evaluate the effects of inflammation on TEE and physical activity energy expenditure in hemodialysis (HD) patients.Design: This was a cross-sectional study.Setting: This study was conducted from Hopital Edouard Herriot, Lyon, France.Patients: This study included 24 HD patients and 18 healthy subjects.Main Outcome Measure: TEE and step counts were measured over a 7-day period by the SenseWear Pro2 Armband in 24 HD patients (15 patients with C-reactive protein,5 mg/L, aged 67.0 +/- 6 14.7 years, and 9 with C-reactive protein >5 mg/L, aged 69.0 +/- 6 18.0 years) and compared with 18 healthy subjects (62.3 +/- 6 15.3 years).Results: Mean estimated TEE measured with SenseWear Pro2 Armband was significantly lower (25.5 +/- 4.1 kcal/kg/day) in patients with inflammation when compared with those without inflammation (32.0 +/- 6.7 kcal/kg/day) and with healthy subjects (31.8 +/- 6 7.0 kcal/kg/day) (P = .012). There was a difference in the physical activity (step counts) between patient groups (P < .05). Healthy subjects and patients without inflammation walked more (8,107 +/- 5,419 and 6,016 +/- 3,752 steps/day, respectively) as compared with patients with inflammation (2,801 +/- 2,754 steps/day, P = .001).Conclusion: Our findings suggest that patients with inflammation have a lower TEE when compared with healthy subjects and patients without inflammation. TEE is influenced by physical activity because patients with inflammation appear to be less active. (C) 2011 by the National Kidney Foundation, Inc. All rights reserved.

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simio-ovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.