Humans and other mammals are limited in their natural abilities to regenerate lost body parts. By contrast, many salamanders are highly regenerative and can spontaneously replace lost limbs even as ...adults. Because salamander limbs are anatomically similar to human limbs, knowing how they regenerate should provide important clues for regenerative medicine. Although interest in understanding the mechanics of this process has never wavered, until recently researchers have been vexed by seemingly impenetrable logistics of working with these creatures at a molecular level. Chief among the problems has been the very large size of salamander genomes, and not a single salamander genome has been fully sequenced to date. Recently the enormous gap in sequence information has been bridged by approaches that leverage mRNA as the starting point. Together with functional experimentation, these data are rapidly enabling researchers to finally uncover the molecular mechanisms underpinning the astonishing biological process of limb regeneration.
The salamander experimental toolset has now largely caught up with the interest in understanding limb regeneration, finally allowing precise experimentation at a cellular and molecular level.
A huge amount of transcript data has emerged from which to gather clues about how limb regeneration occurs.
Differential gene expression analysis has enabled the identification of transcripts that are highly enriched, as well as highly repressed, in key tissues required for limb regeneration. These are prime starting points for hypothesis generation and functional experimentation.
Several genes whose involvement would not have been predicted by candidate gene approaches have now been implicated in limb regeneration, underscoring the need to take unbiased approaches to gene discovery.
De novo transcriptomes and reference-tissue sequence data are important new resources for the field.
Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly ...understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.
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•Creation of a transcriptome with near-complete sequence data for 88% of axolotl genes•Expression analyses identify tissue-enriched transcripts for key tissues•The RNA-binding protein cirbp plays a cytoprotective role in limb regeneration•Knockdown and overexpression of kazald1 in blastema cells impair limb regeneration
Discovery of genes driving axolotl limb regeneration has been challenging, due to limited genomic resources. Bryant et al. have created a transcriptome with near-complete sequence information for most axolotl genes, identified transcriptional profiles that distinguish blastemas from differentiated limb tissues, and uncovered functional roles for cirbp and kazald1 in limb regeneration.
Malignant gliomas metastasize throughout the brain by infiltrative cell migration into peritumoral areas. Invading cells undergo profound changes in cell shape and volume as they navigate ...extracellular spaces along blood vessels and white matter tracts. Volume changes are aided by the concerted release of osmotically active ions, most notably K(+) and Cl(-). Their efflux through ion channels along with obligated water causes rapid cell shrinkage. Suitable ionic gradients must be established and maintained through the activity of ion transport systems. Here, we show that the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1) provides the major pathway for Cl(-) accumulation in glioma cells. NKCC1 localizes to the leading edge of invading processes, and pharmacologic inhibition using the loop diuretic bumetanide inhibits in vitro Transwell migration by 25% to 50%. Short hairpin RNA knockdowns of NKCC1 yielded a similar inhibition and a loss of bumetanide-sensitive cell volume regulation. A loss of NKCC1 function did not affect cell motility in two-dimensional assays lacking spatial constraints but manifested only when cells had to undergo volume changes during migration. Intracranial implantation of human gliomas into severe combined immunodeficient mice showed a marked reduction in cell invasion when NKCC1 function was disrupted genetically or by twice daily injection of the Food and Drug Administration-approved NKCC1 inhibitor Bumex. These data support the consideration of Bumex as adjuvant therapy for patients with high-grade gliomas.
Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer ...selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.
Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a ...reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into ...severe combined immunodeficient mice. Within 14-18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system x(c)(-) cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration-approved drug that blocks system x(c)(-). We found that acute administration of SAS at concentrations equivalent to those used to treat Crohn's disease in humans reduced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when ...combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.
We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific ...RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.
Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been ...extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration.
Empathy is an important human ability associated with successful social interaction. It is currently unclear how to optimally measure individual differences in empathic processing. Although the Big ...Five model of personality is an effective model to explain individual differences in human experience and behavior, its relation to measures of empathy is currently not well understood. Therefore, the present study was designed to investigate the relationship between the Big Five personality concept and two commonly used measures for empathy Empathy Quotient (EQ), Interpersonal Reactivity Index (IRI) in four samples from China, Germany, Spain, and the United States of America. This approach was designed to advance the way the Big Five personality model can be used to measure empathy. We found evidence of medium effect sizes for associations between personality and empathy, with agreeableness and conscientiousness as the most important predictors of affective and cognitive empathy (measured by the respective IRI subscales) as well as for a one-dimensional empathy score (measured by the EQ). Empathy in a fictional context was most closely related to openness to experience while personal distress was first of all related to neuroticism. In terms of culture, we did not observe any distinct pattern concerning cultural differences. These results support the cross-cultural applicability of the EQ and the IRI and indicate structurally similar associations between personality and empathy across cultures.
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