Unique Alterations of an Ultraconserved Non-Coding Element in the 3 ' UTR of ZIC2 in Holoprosencephaly

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.

Prediction of retail beef yield, trim fat and proportion of high-valued cuts in Nellore cattle using ultrasound live measurements

The objective of this study was to develop equations to predict retail product and fat trim (weights and percentages) for Nellore (Bos indicus) cattle. Live ultrasound measurements of the longissimus muscle area, backfat thickness at the 12th rib and rump fat depth and shrunk body weight were obtained from 218 Nellore steers to predict weights and percentages of carcass retail product, pistola retail product and fat trimmings. After slaughter, carcasses were deboned and weighed and percentages of retail cuts were obtained directly. Measurements taken directly in the carcasses explained 97% and 36% of variation in carcass retail product weight and percentage, and 94% and 36% of variation in pistola retail weight and percentage, respectively. Live measurements explained 93% of carcass retail product weight and 39% of carcass retail product percentage. Lower accuracies were observed for pistola retail product weight (R-2=0.87) and percentage (R-2=0.33). Accuracies for fat trimmings weight and percentage were 79% and 55%, respectively. Ultrasound rump fat thickness showed greater correlations with retail product and fat trimmings (weights and percentages) when compared with ultrasound backfat thickness. The weight and percentage of retail products and of trimmable fat can be estimated in Nellore steers from live animal measurements, with similar accuracy to equations developed based on carcass measurements obtained at slaughter.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

A crucial role for IL-6 in the CNS of rats during fever induced by the injection of live E. coli

Interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6 have been established as important mediators of fever induced by lipopolysaccharide (LPS) from Gram-negative bacteria. Whether these pro-inflammatory cytokines are also important in mediating fever induced by live bacteria remains less certain. We therefore investigated the following: (1) the synthesis of TNF-alpha, IL-1 beta, and IL-6 during E. coli-induced fever and (2) the effect of blocking the action of cytokines within the brain on E. coli-induced fever. Body or tail skin temperature (bT or Tsk, respectively) was measured by biotelemetry or telethermometry, every 30 min, during 6 or 24 h. Depending on the number of colony-forming units (CFU) injected i.p., administration of E. coli induced a long-lasting increase in bT of male Wistar rats. The duration of fever did not correlate with the number of CFU found in peritoneal cavity or blood. Because 2.5 x 10(8) CFU induced a sustained fever without inducing a state of sepsis/severe infection, this dose was used in subsequent experiments. The E. coli-induced increase in bT was preceded by a decrease in Tsk, reflecting a thermoregulatory response. TNF-alpha, IL-1 beta, and IL-6 were detected at 3 h in serum of animals injected i.p. with E. coli. In the peritoneal exudates, TNF-alpha, IL-1 beta, and IL-6 were detected at 0.5 and 3 h after E. coli administration. Moreover, both IL-1 beta and IL-6, but not TNF-alpha, were found in the cerebrospinal fluid (CSF) and hypothalamus of animals injected with E. coli. Although pre-treatment (i.c.v., 2 mu l, 15 min before) with anti-IL-6 antibody (anti-IL-6, 5 mu g) reduced E. coli-induced fever, pre-treatment with either IL-1 receptor antagonist (IL-1ra, 200 mu g) or soluble TNF receptor I (sTNFRI, 500 ng) had no effect on the fever response. In conclusion, replicating E. coli promotes an integrated thermoregulatory response in which the central action of IL-6, but not IL-1 and TNF, appears to be important.

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naive patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Bilayer segmentation of live video in uncontrolled environments for background substitution: an overview and main challenges

Bilayer segmentation of live video in uncontrolled environments is an essential task for home applications in which the original background of the scene must be replaced, as in videochats or traditional videoconference. The main challenge in such conditions is overcome all difficulties in problem-situations (e. g., illumination change, distract events such as element moving in the background and camera shake) that may occur while the video is being captured. This paper presents a survey of segmentation methods for background substitution applications, describes the main concepts and identifies events that may cause errors. Our analysis shows that although robust methods rely on specific devices (multiple cameras or sensors to generate depth maps) which aid the process. In order to achieve the same results using conventional devices (monocular video cameras), most current research relies on energy minimization frameworks, in which temporal and spacial information are probabilistically combined with those of color and contrast.

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Androgen responsive intronic non-coding RNAs

Abstract Background Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. Results Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5–5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. Conclusion Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Effects of volume resuscitation on splanchnic perfusion in canine model of severe sepsis induced by live Escherichia coli infusion

Abstract Introduction We conducted the present study to investigate whether early large-volume crystalloid infusion can restore gut mucosal blood flow and mesenteric oxygen metabolism in severe sepsis. Methods Anesthetized and mechanically ventilated male mongrel dogs were challenged with intravenous injection of live Escherichia coli (6 × 109 colony-forming units/ml per kg over 15 min). After 90 min they were randomly assigned to one of two groups – control (no fluids; n = 13) or lactated Ringer's solution (32 ml/kg per hour; n = 14) – and followed for 60 min. Cardiac index, mesenteric blood flow, mean arterial pressure, systemic and mesenteric oxygen-derived variables, blood lactate and gastric carbon dioxide tension (PCO2; by gas tonometry) were assessed throughout the study. Results E. coli infusion significantly decreased arterial pressure, cardiac index, mesenteric blood flow, and systemic and mesenteric oxygen delivery, and increased arterial and portal lactate, intramucosal PCO2, PCO2 gap (the difference between gastric mucosal and arterial PCO2), and systemic and mesenteric oxygen extraction ratio in both groups. The Ringer's solution group had significantly higher cardiac index and systemic oxygen delivery, and lower oxygen extraction ratio and PCO2 gap at 165 min as compared with control animals. However, infusion of lactated Ringer's solution was unable to restore the PCO2 gap. There were no significant differences between groups in mesenteric oxygen delivery, oxygen extraction ratio, or portal lactate at the end of study. Conclusion Significant disturbances occur in the systemic and mesenteric beds during bacteremic severe sepsis. Although large-volume infusion of lactated Ringer's solution restored systemic hemodynamic parameters, it was unable to correct gut mucosal PCO2 gap.

Neural coding tools, based on Information Theory, applied to discrete time series: from electrophysiology to neuroethology

This work is supported by Brazilian agencies Fapesp, CAPES and CNPq

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve) = 0.8 (95% CI = 0.64–0.89) and AUC = 0.90 (95% CI = 0.75–0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.