Project description:Purpose: provide evidence that RNA-seq can add information to transcriptome profiling already discovered by other technologies for atopic dermatitis Methods: mRNA profiles of 20 atopic dermatitis were analyzed to compare lesional and non-lesional skin, then transcriptomes found by reads were compared to Microarray and RT-PCR Results:RNA-seq provided complementary genes to AD transcriptome IL-36 and TREM-1 Conclusions: Our study represents the first analysis of lesional AD tissue by RNA-seq and comparison to microarray and RT-PCR
Project description:A protein's amino acid sequence dictates the folds and final structure the macromolecule will form. We propose that by identifying critical residues in a protein's atomic structure, we can select a critical stability framework within the protein structure essential to proper protein folding. We use global computational mutagenesis based on the unfolding mutation screen to test the effect of every possible missense mutation on the protein structure to identify the residues that cannot tolerate a substitution without causing protein misfolding. This method was tested using molecular dynamics to simulate the stability effects of mutating critical residues in proteins involved in inherited disease, such as myoglobin, p53, and the 15th sushi domain of complement factor H. In addition we prove that when the critical residues are in place, other residues may be changed within the structure without a stability loss. We validate that critical residues are conserved using myoglobin to show that critical residues are the same for crystal structures of 6 different species and comparing conservation indices to critical residues in 9 eye disease-related proteins. Our studies demonstrate that by using a selection of critical elements in a protein structure we can identify a critical protein stability framework. The frame of critical residues can be used in genetic engineering to improve small molecule binding for drug studies, identify loss-of-function disease-causing missense mutations in genetics studies, and aide in identifying templates for homology modeling.
Project description:Pulmonary arterial hypertension (PAH) describes a rare, progressive vascular disease caused by the obstruction of pulmonary arterioles, typically resulting in right heart failure. Whilst PAH most often manifests in adulthood, paediatric disease is considered to be a distinct entity with increased morbidity and often an unexplained resistance to current therapies. Recent genetic studies have substantially increased our understanding of PAH pathogenesis, providing opportunities for molecular diagnosis and presymptomatic genetic testing in families. However, the genetic architecture of childhood-onset PAH remains relatively poorly characterised. We sought to investigate a previously unsolved paediatric cohort (n = 18) using whole exome sequencing to improve the molecular diagnosis of childhood-onset PAH. Through a targeted investigation of 26 candidate genes, we applied a rigorous variant filtering methodology to enrich for rare, likely pathogenic variants. This analysis led to the detection of novel PAH risk alleles in five genes, including the first identification of a heterozygous ATP13A3 mutation in childhood-onset disease. In addition, we provide the first independent validation of BMP10 and PDGFD as genetic risk factors for PAH. These data provide a molecular diagnosis in 28% of paediatric cases, reflecting the increased genetic burden in childhood-onset disease and highlighting the importance of next-generation sequencing approaches to diagnostic surveillance.
Project description:Studies investigating the prevalence, cause, and consequence of multiple sclerosis (MS) fatigue typically use single measures that implicitly assume symptom-stability over time, neglecting information about if, when, and why severity fluctuates. We aimed to examine the extent of moment-to-moment and day-to-day variability in fatigue in relapsing-remitting MS and healthy individuals, and identify daily life determinants of fluctuations. Over 4 weekdays, 76 participants (38 relapsing-remitting MS; 38 controls) recruited from multiple sites provided real-time self-reports six times daily (n = 1661 observations analyzed) measuring fatigue severity, stressors, mood, and physical exertion, and daily self-reports of sleep quality. Fatigue fluctuations were evident in both groups. Fatigue was highest in relapsing-remitting MS, typically peaking in late-afternoon. In controls, fatigue started lower and increased steadily until bedtime. Real-time stressors and negative mood were associated with increased fatigue, and positive mood with decreased fatigue in both groups. Increased fatigue was related to physical exertion in relapsing-remitting MS, and poorer sleep quality in controls. In relapsing-remitting MS, fatigue fluctuates substantially over time. Many daily life determinants of fluctuations are similar in relapsing-remitting MS and healthy individuals (stressors, mood) but physical exertion seems more relevant in relapsing-remitting MS and sleep quality most relevant in healthy individuals.
Project description:Selenium (Se) is an essential mineral element for animals and humans. Cardamine hupingshanensis (Brassicaceae), found in the Wuling mountain area of China, has been identified as a novel Se hyperaccumulator plant. However, the mechanism for selenium tolerance in Cardamine plants remains unknown. In this study, two cDNA libraries were constructed from seedlings of C. hupingshanensis treated with selenite. Approximately 100 million clean sequencing reads were de novo assembled into 48,989 unigenes, of which 39,579 and 33,510 were expressed in the roots and leaves, respectively. Biological pathways and candidate genes involved in selenium tolerance mechanisms were identified. Differential expression analysis identified 25 genes located in four pathways that were significantly responsive to selenite in C. hupingshanensis seedlings. The results of RNA sequencing (RNA-Seq) and quantitative real-time PCR (RT-qPCR) confirmed that storage function, oxidation, transamination and selenation play very important roles in the selenium tolerance in C. hupingshanensis. Furthermore, a different degradation pathway synthesizing malformed or deformed selenoproteins increased selenium tolerance at different selenite concentrations. This study provides novel insights into the mechanisms of selenium tolerance in a hyperaccumulator plant, and should serve as a rich gene resource for C. hupingshanensis.
Project description:Ticks, as obligate blood-feeding arthropod vectors of pathogenic viruses, bacteria, protozoa and helminths, are responsible for prevalent tick-borne diseases (TBDs) worldwide. This arthropod constitutes the second most common that transmit pathogens among humans, after mosquitoes, and the first vector in domestic animals. Vaccines constitute the safest and more effective approach to control tick and TBDs, but this is in constant research to identify new antigens and improve vaccines formulations. The tick antigen Subolesin is a well-known vaccine protective antigen with a highly conserved sequence at both gene and protein levels in the Ixodidae and among arthropods and vertebrates. In this study, RNAseq and proteomic analyses were carried out in wild type and Subolesin knockdown tick ISE6 cells in order to identify and characterize the functional implications of Subolesin in tick cells, demonstrating once again the importance of this antigen in vaccine development against tick and TBDs.
Project description:Physiological trait variation underlies health, responses to global climate change, and ecological performance. Yet, most physiological traits are complex, and we have little understanding of the genes and genomic architectures that define their variation. To provide insight into the genetic architecture of physiological processes, we related physiological traits to heart and brain mRNA expression using a weighted gene co-expression network analysis. mRNA expression was used to explain variation in six physiological traits (whole animal metabolism (WAM), critical thermal maximum (CTmax), and four substrate specific cardiac metabolic rates (CaM)) under 12 °C and 28 °C acclimation conditions. Notably, the physiological trait variations among the three geographically close (within 15 km) and genetically similar F. heteroclitus populations are similar to those found among 77 aquatic species spanning 15-20° of latitude (~ 2,000 km). These large physiological trait variations among genetically similar individuals provide a powerful approach to determine the relationship between mRNA expression and heritable fitness related traits unconfounded by interspecific differences. Expression patterns explained up to 82% of metabolic trait variation and were enriched for multiple signaling pathways known to impact metabolic and thermal tolerance (e.g., AMPK, PPAR, mTOR, FoxO, and MAPK) but also contained several unexpected pathways (e.g., apoptosis, cellular senescence), suggesting that physiological trait variation is affected by many diverse genes.
Project description:In the last 20 years, the use of electron paramagnetic resonance (EPR) has made a pronounced and lasting impact in the field of structural biology. The advantage of EPR spectroscopy over other structural techniques is its ability to target even minor conformational changes in any biomolecule or macromolecular complex, independent of its size or complexity, or whether it is in solution or in the cell during a biological or chemical reaction. Here, we focus on the use of EPR spectroscopy to study transmembrane transport and transcription mechanisms. We discuss experimental and analytical concerns when referring to studies of two biological reaction mechanisms, namely, transfer of copper ions by the human copper transporter hCtr1 and the mechanism of action of the Escherichia coli copper-dependent transcription factor CueR. Last, we elaborate on future avenues in the field of EPR structural biology.
Project description:Polymerisation of clathrin is a key process that underlies clathrin-mediated endocytosis. Clathrin-coated vesicles are responsible for cell internalization of external substances required for normal homeostasis and life -sustaining activity. There are several hypotheses describing formation of closed clathrin structures. According to one of the proposed mechanisms cage formation may start from a flat lattice buildup on the cellular membrane, which is later transformed into a curved structure. Creation of the curved surface requires rearrangement of the lattice, induced by additional molecular mechanisms. Different potential mechanisms require a modeling framework that can be easily modified to compare between them. We created an extendable rule-based model that describes polymerisation of clathrin molecules and various scenarios of cage formation. Using Global Sensitivity Analysis (GSA) we obtained parameter sets describing clathrin pentagon closure and the emergence/production and closure of large-size clathrin cages/vesicles. We were able to demonstrate that the model can reproduce budding of the clathrin cage from an initial flat array.
Project description:Accurate and precise annotation of the 3Ⲡuntranslated regions (3ⲠUTRs) is critical in understanding how mRNA stability, localization and translational efficiency are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here we describe a novel method, PAPERCLIP (Poly(A) binding Protein-mediated mRNA 3´End Retrieval by CrossLinking ImmunoPrecipitation), which shows high specificity for the mRNA 3Ⲡends and compares favorably to existing 3Ⲡend mapping methods including direct RNA sequencing in performance. PAPERCLIP protocol and RNA-Seq on human cell lines and mouse whole brain cortex.