Study of Heparin in Intestinal Ischemia and Reperfusion in Rats: Morphologic and Functional Evaluation

To study whether treatment with heparin (HEP) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with HEP (100 U/kg intravenously) or saline solution (SS) before I (60 min), which was produced by occlusion of the superior mesenteric artery, and R (120 min). After I or I/R, we mounted 2-cm jejunal segment in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl, using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the I + HEP and the I/R + HEP groups, but reduced in the I + SS and the I/R + SS groups. the jejunal enteric nerves were damaged in the I + SS and the I/R + SS, but not in the I + HEP and the I/R + HEP cohorts. These results suggested that HEP attenuated intestinal dysfunction caused by I and I/R.Universidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morphol, BR-04023900 São Paulo, BrazilFed Univ Great Dourados, Sch Med, Dourados, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morphol, BR-04023900 São Paulo, BrazilWeb of Scienc

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in “non-model” species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies

Performance of Lotus corniculatus L. genotypes submitted to cutting interval: subsidies to a breeding program

The objective of this work was to evaluate the response of five birdsfoot trefoil populations (Lotus corniculatus L.), selected under severe and intense grazing (P38, P37, P9 and P3) or cutting (population Corte), one rhizomatous population from Morocco and two cultivars, São Gabriel (Brazil) and ARS 2620 (USA), to different cutting intervals (20 and 40 days). The trial was carried out in a greenhouse for 224 days. The plants were submitted to the treatments during four months, when it was made four and two cuttings for the 20 and 40 day intervals, respectively. In the following months, aerial part of the plants was totally removed and after 70 days of regrowth, evaluation of roots and aerial section were performed. Analysis of variance and multivariate analysis was performed, obtaining the Mahalanobis distance (Md), dendogram by UPGMA method and the relative contribution of the characters for genetic divergence. There was no genotype × cutting interval interaction and the genotypes presented a reduction on dry matter yield of roots, crown and aerial sections, number of stems and plant height when submitted to frequent cuttings. The most divergent genotypes were Marrocos and P9 (Md = 108.7) and the most similar ones were São Gabriel and P37 (Md = 7.8). The results suggest exclusion of the population P9 because of its weak performance and the utilization of the populations Corte and P37 as progenitors in the birdsfoot trefoil breeding program. Root dry matter accumulation and plant height were the characters that contributed most to genetic divergence and they can be used for selection works

Treatment of refractory epilepsy with natalizumab in a patient with multiple sclerosis. Case report

Background. Multiple sclerosis (MS) is considered an autoimmune disease of the central nervous system and therapeutic inhibition of leukocyte migration with natalizumab, an anti-alpha4 integrin antibody, is highly effective in patients with MS. Recent studies performed in experimental animal models with relevance to human disease suggested a key role for blood-brain barrier damage and leukocyte trafficking mechanisms also in the pathogenesis of epilepsy. In addition, vascular alterations and increased leukocyte accumulation into the brain were recently documented in patients with refractory epilepsy independently on the disease etiology. Case report. Here we describe the clinical course of a 24-year-old patient with MS in whom abrupt tonic-clonic generalized seizures manifested at disease onset. Although MS had a more favorable course, treatment with glatiramer acetate and antiepileptic drugs for 7 years had no control on seizure generation and the patient developed severe refractory epilepsy. Interestingly, generalized seizures preceded new MS relapses suggesting that seizure activity may contribute to MS worsening creating a positive feedback loop between the two disease conditions. Notably, treatment with natalizumab for 12 months improved MS condition and led to a dramatic reduction of seizures. Conclusion. Our case report suggests that inhibition of leukocyte adhesion may represent a new potential therapeutic approach in epilepsy and complement the traditional therapy with anti-epileptic drugs

Parallel evolution and adaptation to environmental factors in a marine flatfish: implications for fisheries and aquaculture management of the turbot (<i>Scophthalmus maximus</i>)

Unraveling adaptive genetic variation represents, in addition to the estimate of population demographic parameters, a cornerstone for the management of aquatic natural living resources, which, in turn, represent the raw material for breeding programs. The turbot (Scophthalmus maximus) is a marine flatfish of high commercial value living on the European continental shelf. While wild populations are declining, aquaculture is flourishing in southern Europe. We evaluated the genetic structure of turbot throughout its natural distribution range (672 individuals; 20 populations) by analyzing allele frequency data from 755 single nucleotide polymorphism discovered and genotyped by double‐digest RAD sequencing. The species was structured into four main regions: Baltic Sea, Atlantic Ocean, Adriatic Sea, and Black Sea, with subtle differentiation apparent at the distribution margins of the Atlantic region. Genetic diversity and effective population size estimates were highest in the Atlantic populations, the area of greatest occurrence, while turbot from other regions showed lower levels, reflecting geographical isolation and reduced abundance. Divergent selection was detected within and between the Atlantic Ocean and Baltic Sea regions, and also when comparing these two regions with the Black Sea. Evidence of parallel evolution was detected between the two low salinity regions, the Baltic and Black seas. Correlation between genetic and environmental variation indicated that temperature and salinity were probably the main environmental drivers of selection. Mining around the four genomic regions consistently inferred to be under selection identified candidate genes related to osmoregulation, growth, and resistance to diseases. The new insights are useful for the management of turbot fisheries and aquaculture by providing the baseline for evaluating the consequences of turbot releases from restocking and farming

Identification and Characterization of the Lamprey High-Mobility Group Box 1 Gene

High-mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. We identified a homolog of HMGB1 in the Japanese lamprey (Lampetra japonica). The Lampetra japonica HMGB1 gene (Lj-HMGB1) has over 70% sequence identity with its homologs in jawed vertebrates. Despite the reasonably high sequence identity with other HMGB1 proteins, Lj-HMGB1 did not group together with these proteins in a phylogenetic analysis. We examined Lj-HMGB1 expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with lipopolysaccharide (LPS) and concanavalin A (ConA). Lj-HMGB1 was initially expressed at a higher level in the heart, but after treatment with LPS and ConA only the gills demonstrated a significant up-regulation of expression. The recombinant Lj-HMGB1 (rLj-HMGB1) protein bound double-stranded DNA and induced the proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. We further revealed that Lj-HMGB1 was able to induce the production of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator, in activated human acute monocytic leukemia cells. These results suggest that lampreys use HMGB1 to activate their innate immunity for the purpose of pathogen defense

Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures

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Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 beta

<p>Abstract</p> <p>Objective</p> <p>Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K⁺ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression.</p> <p>Methods</p> <p>We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (<it>n</it> = 64), comparing the expression in tumor patients with (<it>n</it> = 38) and without epilepsy (<it>n</it> = 26).</p> <p>Results</p> <p>Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1).</p> <p>Conclusions</p> <p>Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the inflammatory cytokine IL-1β.</p