Diagnostic value of lipopolysaccharide-binding protein and procalcitonin for sepsis diagnosis in forensic pathology.

The aims of this study were twofold. The first was to investigate the diagnostic performance of two biochemical markers, procalcitonin (PCT) and lipopolysaccharide-binding protein (LBP), considering each individually and then combined, for the postmortem diagnosis of sepsis. We also tested the usefulness of pericardial fluid for postmortem LBP determination. Two study groups were formed, a sepsis-related fatalities group of 12 cases and a control group of 30 cases. Postmortem native CT scans, autopsy, histology, neuropathology, and toxicology as well as other postmortem biochemical investigations were performed in all cases. Microbiological investigations were also carried out in the septic group. Postmortem serum PCT and LBP levels differed between the two groups. Both biomarkers, individually considered, allowed septic states to be diagnosed, whereas increases in both postmortem serum PCT and LBP levels were only observed in cases of sepsis. Similarly, normal PCT and LBP values in postmortem serum were identified only in non-septic cases. Pericardial fluid LBP levels do not correlate with the presence of underlying septic states. No relationship was observed between postmortem serum and pericardial fluid LBP levels in either septic or non-septic groups, or between pericardial fluid PCT and LBP levels.

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

Molecular and Antigenic Characterization of the Leishmania (Viannia) panamensis Kinetoplastid Membrane Protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Allelic Diversity at the Merozoite Surface Protein-1 (MSP-1) Locus in Natural Plasmodium falciparum Populations: a Brief Overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed

Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase. After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters. The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA. Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons. Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation. The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF). However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action.

A long-term intake of a protein hydrolysate seems to increase the risk of encephalopathy in mice infected with Schistosoma mansoni

Previous investigations showed that Schistosoma mansoni infection aggravates protein malabsorption in undernourished mice and this can be reverted by administration of casein hydrolysate. The present study was undertaken to evaluate the effects of ingestion of casein hydrolysate for long periods. Albino Swiss mice were divided into eight groups. Diets contained 5% (undernourished ) or 20% (controls) casein levels. For each group there were sub-groups ingesting whole or hydrolysed casein for 12 weeks. Infection with S. mansoni developed in half of the animals under each diet. All undernourished mice developed malabsorption. Low albuminemia was detected in infected animals independently of the protein level in the diet. However, albuminemia was lower in infected controls than in undernourished non-infected mice, suggesting a deficient liver protein synthesis. Infected mice fed on a 20% protein hydrolysed diet exhibited low weight gain and high mortality rates. On the other hand, non-infected mice ingesting the same diet had the highest body weights. We are investigating the hypothesis that infected mice, even when fed normal diets, are unable to metabolise large amounts of amino acids due to the liver lesions related to schistosomiasis and as a result die of hepatic coma. In some of them, the excessive accumulation of ammonia in the blood enhances the outcome of an encephalopathy.

Protein intake and exercise for optimal muscle function with aging: recommendations from the ESPEN Expert Group.

The aging process is associated with gradual and progressive loss of muscle mass along with lowered strength and physical endurance. This condition, sarcopenia, has been widely observed with aging in sedentary adults. Regular aerobic and resistance exercise programs have been shown to counteract most aspects of sarcopenia. In addition, good nutrition, especially adequate protein and energy intake, can help limit and treat age-related declines in muscle mass, strength, and functional abilities. Protein nutrition in combination with exercise is considered optimal for maintaining muscle function. With the goal of providing recommendations for health care professionals to help older adults sustain muscle strength and function into older age, the European Society for Clinical Nutrition and Metabolism (ESPEN) hosted a Workshop on Protein Requirements in the Elderly, held in Dubrovnik on November 24 and 25, 2013. Based on the evidence presented and discussed, the following recommendations are made (a) for healthy older people, the diet should provide at least 1.0-1.2 g protein/kg body weight/day, (b) for older people who are malnourished or at risk of malnutrition because they have acute or chronic illness, the diet should provide 1.2-1.5 g protein/kg body weight/day, with even higher intake for individuals with severe illness or injury, and (c) daily physical activity or exercise (resistance training, aerobic exercise) should be undertaken by all older people, for as long as possible.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

No Role for Cerebrospinal Fluid Myelin Basic Protein Levels in Patients Treated for Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Central nervous system prophylaxis of childhood acute lymphoblastic leukemia has dropped rates of relapses but has been associated with neurotoxicity and imaging abnormalities. Predictors of neurotoxicity are lacking, because of inconsistency between clinical symptoms and imaging. Some have suggested that cerebrospinal fluid myelin basic protein (MBP) levels to be of potential interest. A retrospective analysis of MBP levels in correlation with clinical and radiologic 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 neurologic examinations in 34 patients and 69 neuroradiologic investigations in 27 patients. Fifty-two patients had abnormal MBP levels. Radiologic 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 radiologic anomalies or with no radiologic 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 acute lymphoblastic leukemia patients.