Bioinformatic Tools and Guidelines for the Design of Fluorescence In Situ Hybridization Probes.

Fluorescence in situ hybridization (FISH) is a well-established technique that allows the detection of microorganisms in diverse types of samples (e.g., clinical, food, environmental samples, and biofilm communities). The FISH probe design is an essential step in this technique. For this, two strategies can be used, the manual form based on multiple sequence alignment to identify conserved regions and programs/software specifically developed for the selection of the sequence of the probe. Additionally, databases/software for the theoretical evaluation of the probes in terms of specificity, sensitivity, and thermodynamic parameters (melting temperature and Gibbs free energy change) are used. The purpose of this chapter is to describe the essential steps and guidelines for the design of FISH probes (e.g., DNA and Nucleic Acid Mimic (NAM) probes), and its theoretical evaluation through the application of diverse bioinformatic tools.

Uncommon Movement Disorders in Chronic Hepatic Disease with Response to Rifaximin.

Background: Chronic hepatic disease can present with extrapyramidal symptoms. We describe two cases that presented with highly unusual movement disorders: ballism and gait freezing. Case report: Patient 1 is a 42-year-old man with previous episodes of hepatic encephalopathy (HE) who presented with upper limb dystonia and generalized chorea that progressed to ballism. Patient 2 is a 55-year-old woman who presented with pronounced gait freezing. In both patients, features of HE and acquired hepatocerebral degeneration coexisted. They improved markedly, though transiently, with rifaximin. Discussion: Ammonia-reducing treatments should be considered in patients presenting with movement disorders due to chronic liver disease.

Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a "spotwelding" activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.

Proinflammatory and anti-inflammatory cytokines in the CSF of patients with Alzheimer's disease and their correlation with cognitive decline.

Cumulative data suggest that neuroinflammation plays a prominent role in Alzheimer's disease (AD) pathogenesis. The purpose of this work was to assess if patients with AD present a specific cerebrospinal fluid (CSF) cytokine profile and if it correlates to disease progression. We determined the levels of 27 cytokines in CSF of patients with AD and compared them with patients with frontotemporal dementia and nondemented controls. In addition, we correlated the cytokine levels with cognitive status and disease progression after 12 months. Patients with AD had higher levels of proinflammatory and anti-inflammatory cytokines (eotaxin, interleukin [IL]-1ra, IL-4, IL-7, IL-8, IL-9, IL-10, IL-15, granulocyte colony-stimulating factor, monocyte chemotactic protein 1, platelet-derived growth factor, tumor necrosis factor alfa) compared to nondemented controls. There was a negative correlation between the disease progression and the levels of several cytokines (IL-1ß, IL-4, IL-6, IL-9, IL-17A, basic fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon gamma, macrophage inflammatory proteins-1ß). To the best of our knowledge, this is the first study reporting a "protective" role of the upregulation of specific intrathecal cytokine levels in AD. This finding supports that a fine "rebalancing" of the immune system represents a new target in AD therapeutic approach.

Home blood pressure normalcy in non-European adolescents.

OBJECTIVE: Evidence on normal range of home blood pressure (BP) in adolescents relies on only one European study. This study aims to investigate the normal range of home BP in a healthy non-European population of adolescents. METHODS: Cross-sectional study with a representative sample of secondary school students (12-17 years) from a Brazilian state capital. Adolescents' heights were classified in percentiles according to age and sex. Height percentiles were divided in 50th or less or more than 50th percentile. The home BP protocol included two day-time and two evening-time measurements over 6 days. Exams were considered valid with at least 12 measurements. RESULTS: A total of 1024 adolescents were included, mean age 15.21 ±â€Š1.61 years, mostly women (52.4%), from public schools (68.4%) and nonwhite (51.3%). The 50th (midpoint of distribution) and the 95th percentile (upper normal limit) for home SBP and DBP in adolescents are provided by sex, age and height percentiles. There was a marked increase in the estimated 95th percentile for home SBP with increasing age in men for both height percentiles examined (16 mmHg for ≤50th percentile and 14.5 mmHg for >50th percentile) and less so for home DBP (1 and 5 mmHg, respectively). In women, the 95th percentile increase with age was less significant for SBP and similar for DBP when compared with men in the two height percentiles evaluated (6/2 mmHg for ≤50th percentile and 4/4 mmHg for >50th percentile). CONCLUSION: Reference values for home BP by height percentiles for age and sex in a non-European population of adolescents are provided.

Sympathetic neuron-associated macrophages contribute to obesity by importing and metabolizing norepinephrine.

The cellular mechanism(s) linking macrophages to norepinephrine (NE)-mediated regulation of thermogenesis have been a topic of debate. Here we identify sympathetic neuron-associated macrophages (SAMs) as a population of cells that mediate clearance of NE via expression of solute carrier family 6 member 2 (SLC6A2), an NE transporter, and monoamine oxidase A (MAOA), a degradation enzyme. Optogenetic activation of the sympathetic nervous system (SNS) upregulates NE uptake by SAMs and shifts the SAM profile to a more proinflammatory state. NE uptake by SAMs is prevented by genetic deletion of Slc6a2 or inhibition of the encoded transporter. We also observed an increased proportion of SAMs in the SNS of two mouse models of obesity. Genetic ablation of Slc6a2 in SAMs increases brown adipose tissue (BAT) content, causes browning of white fat, increases thermogenesis, and leads to substantial and sustained weight loss in obese mice. We further show that this pathway is conserved, as human sympathetic ganglia also contain SAMs expressing the analogous molecular machinery for NE clearance, which thus constitutes a potential target for obesity treatment.

Does nutritional status interfere with adolescents' body image perception?

Adolescents' body image (BI) may not match their nutritional status. This study selected representative sample of healthy adolescents aged between 12 and 18 from public and private schools. Anthropometric measures were performed in order to calculate the body mass index (BMI) percentile. The silhouette scale proposed by Childress was used to evaluate BI, making it possible to assess BI satisfaction and BI distortion. The sample was composed of 1168 adolescents with a mean age of 14.7 years; 52.9% were female, 50.9% were fair-skinned, 62.4% had consumed or still consume alcohol and 67% attended public school. Male adolescents presented more overweight and obesity (28.4%) (p<0.05) than the female (17.1%). It was observed that 69.4% were dissatisfied with BI, 91.1% of the obese and 69.8% of those with overweight wished to lose body weight and 82.5% of those underweight wished to gain body weight. BI distortion was identified, since 35% of the adolescents who were underweight did not regard themselves thin, 39.1% of the overweight individuals and 62.1% of the obese did not see themselves in their adequate classifications. Adolescents with overweight/obesity were those who presented higher dissatisfaction with BI, mainly the females. Male individuals presented a greater wish of gaining weight. BI distortion was present in adolescents of all classes of BMI percentile.