Changing genetic paradigms: creating next-generation genetic databases as tools to understand the emerging complexities of genotype/phenotype relationships

Understanding genotype/phenotype relationships has become more complicated as increasing amounts of inter- and intra-tissue genetic heterogeneity have been revealed through next-generation sequencing and evidence showing that factors such as epigenetic modifications, non-coding RNAs and RNA editing can play an important role in determining phenotype. Such findings have challenged a number of classic genetic assumptions including (i) analysis of genomic sequence obtained from blood is an accurate reflection of the genotype responsible for phenotype expression in an individual; (ii) that significant genetic alterations will be found only in diseased individuals, in germline tissues in inherited diseases, or in specific diseased tissues in somatic diseases such as cancer; and (iii) that mutation rates in putative disease-associated genes solely determine disease phenotypes. With the breakdown of our traditional understanding of genotype to phenotype relationships, it is becoming increasingly apparent that new analytical tools will be required to determine the relationship between genotype and phenotypic expression. To this end, we are proposing that next-generation genetic database (NGDB) platforms be created that include new bioinformatics tools based on algorithms that can evaluate genetic heterogeneity, as well as powerful systems biology analysis tools to actively process and evaluate the vast amounts of both genomic and genomic-modifying information required to reveal the true relationships between genotype and phenotype.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Balance between the two kinin receptors in the progression of experimental focal and segmental glomerulosclerosis in mice

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). In order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. The blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease.KEY WORDS: Focal and segmental glomerulosclerosis, Bradykinin receptors, Inflammation, Podocyte, Fibrosis     

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.     

Galactodendritic Phthalocyanine Targets Carbohydrate-Binding Proteins Enhancing Photodynamic Therapy

Photosensitizers (PSs) are of crucial importance in the effectiveness of photodynamic therapy (PDT) for cancer. Due to their high reactive oxygen species production and strong absorption in the wavelength range between 650 and 850 nm, where tissue light penetration is rather high, phthalocyanines (Pcs) have been studied as PSs of excellence. In this work, we report the evaluation of a phthalocyanine surrounded by a carbohydrate shell of sixteen galactose units distributed in a dendritic manner (PcGal₁₆) as a new and efficient third generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UM-UC-3. Here, we define the role of galacto-dendritic units in promoting the uptake of a Pc through interaction with GLUT1 and galectin-1. The photoactivation of PcGal₁₆ induces cell death by generating oxidative stress. Although PDT with PcGal₁₆ induces an increase on the activity of antioxidant enzymes immediately after PDT, bladder cancer cells are unable to recover from the PDT-induced damage effects for at least 72 h after treatment. PcGal₁₆ co-localization with galectin-1 and GLUT1 and/or generation of oxidative stress after PcGal₁₆ photoactivation induces changes in the levels of these proteins. Knockdown of galectin-1 and GLUT1, via small interfering RNA (siRNA), in bladder cancer cells decreases intracellular uptake and phototoxicity of PcGal₁₆. The results reported herein show PcGal₁₆ as a promising therapeutic agent for the treatment of bladder cancer, which is the fifth most common type of cancer with the highest rate of recurrence of any cancer.     

Proteomic Analysis of Gastrocnemius Muscle in Rats with Streptozotocin-Induced Diabetes and Chronically Exposed to Fluoride

Administration of high doses of fluoride (F) can alter glucose homeostasis and lead to insulin resistance (IR). This study determined the profile of protein expression in the gastrocnemius muscle of rats with streptozotocin-induced diabetes that were chronically exposed to F. Male Wistar rats (60 days old) were randomly distributed into two groups of 18 animals. In one group, diabetes was induced through the administration of streptozotocin. Each group (D-diabetic and ND-non-diabetic) was further divided into 3 subgroups each of which was exposed to a different F concentration via drinking water (0 ppm, 10 ppm or 50 ppm F, as NaF). After 22 days of treatment, the gastrocnemius muscle was collected and submitted to proteomic analysis (2D-PAGE followed by LC-MS/MS). Protein functions were classified by the GO biological process (ClueGO v2.0.7+Clupedia v1.0.8) and protein-protein interaction networks were constructed (PSICQUIC, Cytoscape). Quantitative intensity analysis of the proteomic data revealed differential expression of 75 spots for ND0 vs. D0, 76 for ND10 vs.D10, 58 spots for ND50 vs. D50, 52 spots for D0 vs. D10 and 38 spots for D0 vs. D50. The GO annotations with the most significant terms in the comparisons of ND0 vs. D0, ND10 vs. D10, ND50 vs. D50, D0 vs. D10 and D0 vs. D50, were muscle contraction, carbohydrate catabolic processes, generation of precursor metabolites and energy, NAD metabolic processes and gluconeogenesis, respectively. Analysis of subnetworks revealed that, in all comparisons, proteins with fold changes interacted with GLUT4. GLUT4 interacting proteins, such as MDH and the stress proteins HSPB8 and GRP78, exhibited decreased expression when D animals were exposed to F. The presence of the two stress proteins indicates an increase in IR, which might worsen diabetes. Future studies should evaluate whether diabetic animals treated with F have increased IR, as well as which molecular mechanisms are involved.     

Systematic Review of the Efficacy of Cognitive-Behavior Therapy Related Treatments for Victims of Natural Disasters: A Worldwide Problem

Natural disasters can have devastating consequences. Each year, about 225 million people are victims of natural disasters worldwide, and up to 13,5 million of these people can develop post-traumatic stress disorder (PTSD) in the first or second year following the disaster. Cognitive-behavior therapy (CBT) is the first-choice treatment for this disorder. In order to evaluate the efficacy of psychotherapeutic treatment based on cognitive-behavior therapy for people who developed post traumatic stress disorder after natural disasters we conducted a systematic search of published studies. We used the terms reported below in the electronic databases ISI Web of Science, PsycINFO, PubMed, PILOTS and Scopus with no restrictions of language or publication date. Articles that described randomized controlled, non-randomized controlled and non controlled studies on the efficacy of cognitive-behavior therapy for individuals diagnosed with post-traumatic stress disorder after exposure to a natural disaster were eligible for inclusion. The studies were required to use a standardized measure of effectiveness before and after the intervention and have a group of patients who had used cognitive-behavior therapy as the only intervention. Our search identified 820 studies, and 11 were selected for this review. These 11 studies involved 742 subjects, 10 related to earthquakes and 1 to a hurricane. The cognitive-behavior therapy techniques used were various: 7 studies used exposure therapy, 2 studies used problem solving, and the only 2 studies with adolescents used techniques including reconstructions and reprocessing of the traumatic experience. As limitations, the search involved only five electronic databases, no experts in the field were consulted, and the heterogeneity of the findings made it impossible to perform a meta-analysis. The results suggest the efficacy of cognitive-behavior therapy, particularly exposure techniques, for the treatment of post-traumatic stress disorder after earthquakes. However, further studies with stronger methodologies, i.e. randomized-control trials and non-randomized controlled trials, are needed.     

Importance of Cough and M. tuberculosis Strain Type as Risks for Increased Transmission within Households

Rationale

The degree to which tuberculosis (TB) is transmitted between persons is variable. Identifying the factors that contribute to transmission could provide new opportunities for TB control. Transmission is influenced by host, bacterial and environmental factors. However, distinguishing their individual effects is problematic because measures of disease severity are tightly correlated, and assessing the virulence of Mycobacterium tuberculosis isolates is complicated by epidemiological and clinical confounders.

Objectives

To overcome these problems, we investigated factors potentially associated with TB transmission within households.

Methods

We evaluated patients with smear-positive (≥2+), pulmonary TB and classified M. tuberculosis strains into single nucleotide polymorphism genetic cluster groups (SCG). We recorded index case, household contact, and environmental characteristics and tested contacts with tuberculin skin test (TST) and interferon-gamma release assay. Households were classified as high (≥70% of contacts with TST≥10 mm) and low (≤40%) transmission. We used logistic regression to determine independent predictors.

Result

From March 2008 to June 2012, we screened 293 TB patients to enroll 124 index cases and their 731 contacts. There were 23 low and 73 high transmission households. Index case factors associated with high transmission were severity of cough as measured by a visual analog cough scale (VACS) and the Leicester Cough Questionnaire (LCQ), and cavitation on chest radiograph. SCG 3b strains tended to be more prevalent in low (27.3%) than in high (12.5%) transmission households (p = 0.11). In adjusted models, only VACS (p<0.001) remained significant. SCG was associated with bilateral disease on chest radiograph (p = 0.002) and marginally associated with LCQ sores (p = 0.058), with group 3b patients having weaker cough.

Conclusions

We found differential transmission among otherwise clinically similar patients with advanced TB disease. We propose that distinct strains may cause differing patterns of cough strength and cavitation in the host leading to diverging infectiousness. Larger studies are needed to verify this hypothesis.