We have shown that many splicing differences between healthy individuals can be identified using the Exon Array platform. We expect that many more, perhaps approaching the complete map of the splicing eQTL (expression quantitative trait loci) landscape, will be catalogued soon using more sensitive methods, such as deep mRNA sequencing. Most of these splicing differences which we can detect are controlled by polymorphisms in cis-regulatory regions or in the vicinity of an implicated splice-site. These differences between individuals could contribute to phenotypic variationand could either be neutral in their effects, or confer differential susceptibility to complex diseases. A few of our validated events occur in genes which have already been associated with diseases. BCKDHA is related to maple syrup urine disease, type 1a , a rare inherited metabolic disorder which, without a highly controlled diet and close monitoring of blood chemistry, causes progressive neurological damage which can cause vomiting, eating difficulties, irregular breathing, coma or death. Deficiency of the gene alpha-methylacyl-coa racemase (AMACR) is a rare disorder of the fatty acid metabolism which is characterized by neuronal and liver abnormalities  and the gene is considered a useful biomarker for various types of cancer , making it quite interesting that this gene contained the SNP with the most evidence of positive selection. Interleukin 6 (IL6) is an important mediator of fever  and the gene has been associated with osteoporosis  and Kaposi’s sarcoma . MMAB is related to vitamin B12 responsive methylmalonic aciduria , the inability to synthesize adenosylcobalamin, a vitamin B12 derivative, and whose symptoms include metabolic acidosis and retarded development. A SNP in MMAB, which is in linkage disequilibrium with our causative splicing SNP, has recently been associated with HDL cholesterol levels . Surprisingly, all of the above AS events are within protein-coding regions of the genes, making it very likely that these heritable differences contribute to individuals’ predisposition to disorders similar to those caused by inactivation of those genes or to other, more complex, diseases.
Large-scale sequencing efforts have documented extensive geneticvariation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternativesplicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicingacross populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternativesplicing, and regulatory genetics across populations from the broadest points of human migration history yet sampled.
Understanding how geneticvariation affects distinct cellular phenotypes, such as gene ex- pression levels, alternativesplicingand DNA methylation levels, is essential for better un- derstanding of complex diseases and traits. Furthermore, how inter-individual variationof DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously geno- typed for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the corre- lation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression ap- pears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance ofgenetic effects can also be reflected by the ob- servation that allele specific expression differences between individuals dominate over tis- sue-specific effects. Additionally, we discover genetic effects on alternativesplicingand interestingly, a large amount of DNA methylation correlating to alternativesplicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternativesplicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer ofvariation that is more tissue- a11111
Five reports [53–57] indicate that de novo Alu insertions into intronic sequences in antisense orientation and in close proximity to the affected exon (between 19–50 nucleotides) cause the down- stream exon to shift from constitutive splicing to full exon skipping (three cases) or to alternativesplicing (two cases) (Table 1). This effect of Alu elements on adjacent exons may be due to the Alu structure. Alu elements are comprised of two very similar segments, termed left and right arms. When an Alu is located in a gene in the antisense orientation and transcribed it contributes two poly-T stretches to the mRNA precursor. These poly-T regions might act as polypyrimidine tract (PPT) and, in combination with downstream 3 9 and 59 pseudo splice sites, might act as pseudo-exon . Hence, such antisense Alus that act as pseudo-exons might compete with nearby exons for the binding ofsplicing factors. These five cases of de novo Alu insertions imply that Alus located in close proximity to exons might affect splicingof adjacent exons. This and the finding of de-novo Alu insertions that affect splicing imply that this is an on- going evolutionary process, which may result in novel transcripts that are deleterious and inflict genetic diseases. On the other hand, a shift in the splicing pattern from constitutive to alternative might be advantageous in some cases, and could enable testing new mRNA options without eliminating the old ones. Moreover, such a shift could introduce a premature termination codons enabling the expression of truncated proteins at certain needed times or in specific cell types and could be delicately regulated by the levels ofsplicing regulatory proteins [59,60].
important role in cerebral malaria (CM) physiopathology by inducing changes in cerebral endothelial cells, leading to the surface expression of adhesion molecules, thereby enhancing parasitized erythrocyte sequestration [3,4]. When TNF-α is excessively produced, malaria patients suffer from fever, hypoglycemia, bone marrow, depression, coagulopathy, hypergammaglobulinemia, hypotension and rise in serum levels . In a large case control study of Gambian children, a genetic variant of the TNF-α gene promoter region was found to have a relative risk of seven for death or severe neurological sequels due to severe malaria, suggesting that regulatory polymorphisms of cytokine genes affect the outcome of severe infection . However, TNF-α and another cytokine, interferon gamma (IFN-γ) have been shown to govern a new type of protective response against infectious agents in malaria, in addition to causing disease when generated in excess . This dual role (protective and pathogenic) of TNF-α depends on the quantity of TNF-α produced, the time-period over which its production is sustained, the tissue in which it is produced, and on other cytokines that regulate its production [6,7]. Patients infected with the malaria parasite Plasmodium falciparum have higher levels of TNF-α; these levels are directly proportional to the severity of disease . Apart from malaria, the dual role of TNF-α also extends to other infectious diseases, such as leishmaniasis; it is well documented that TNF-α, along with IFN-γ, are over produced in cutaneous and mucosal leishmaniasis .
To overcome these limitations, we have been developing an R application with a graphical inter- face for the integrated analysis of AS from large transcriptomic datasets, namely from The Cancer Genome Atlas (TCGA) project. The tool interactively performs clustering, principal component and other graphically-assisted exploratory analyses. Amongst its innovative aspects are the analysis of vari- ance (which our research shows to be important in the detection of otherwise unnoticed putative targets) and the direct incorporation of clinical features (such as tumour stage or survival) associated with TCGA samples. Interactive visual access to genomic mapping and functional annotation of selected AS events is also incorporated. We have successfully used the application in the revelation of cancer-specific AS signatures and associated novel putative prognostic factors.
Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternativesplicingof several genes has also been correlated with EMT progression, but the extent ofsplicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternativesplicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternativesplicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes ofsplicing factors. The EMT alternativesplicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternativesplicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.
The cohort comprised HIV-infected patients (cases) matched to HIV-uninfected patients (con- trols) on the basis of age, gender, race, and number of medical encounters in a 3:1 ratio. Data were obtained from the Partners HealthCare System (PHS) Research Patient Data Registry (RPDR), a comprehensive database of administrative, billing and electronic health record (EHR) information including inpatient and outpatient encounters for over 4.5 million patients. Patients were eligible to be included as cases if they received care at Brigham and Women’s Hospital or Massachusetts General Hospital between 2005 and 2007. HIV infection was deter- mined by inpatient or outpatient diagnosis of HIV (International Classification of Diseases, 9th Revision, Clinical Modification [ICD-9-CM] codes 042 and all subtypes, V08, and corre- sponding electronic health record codes). Exclusion criteria for both groups included diagnosis of coronary heart disease (CHD) prior to 2008, age <18 years, and death prior to January 1, 2008. The study period spanned the time of the earliest documented clinical encounter through October 31, 2008. This study was approved by the Partners Human Research Committee. Informed consent of study subjects was not obtained. The IRB approval included a waiver of the requirement to obtain informed consent because the risk to study subjects, including risk to privacy, was deemed to be minimal, obtaining informed consent of study subjects was not feasible and the rights and welfare of the subjects would not be adversely affected by the waiver.
In this paper, we will suggest that the intertidal organisms of the North Atlantic offer a rich set of material to test hypotheses of selection along environ- mental gradients. Many of the species in the North Atlantic are recent colonists, having arrived from the Paciﬁc after the opening of the Bering Strait 5 Ma (million years ago), with most arriving during the great invasion 3.5 Ma (Marincovich and Gladenkov 1999). This introduced a number of species onto both coasts of the Atlantic, offering an opportunity to study parallel aspects of ecological and evolutionary adaptations within the same set of species. A particularly attractive aspect of these organisms is the extensive ecological literature describing patterns and processes governing recruitment, reproduction, and community structure. A further strength of these communities is the multiple spatial scales over which clinal variation can be examined: there are parallel gradients spanning latitude, estuaries, and tidal height on both coasts of the North Atlantic. While most intertidal organisms are poor models for geneticand genomic analyses, progress in comparative genomics will facilitate the application of molecular methods to many non-model genetic organ- isms (e.g., Stillman et al. 2006, Teranishi and Stillman 2007). Moreover, statistical methods in population genetics have provided some powerful approaches to the study of adaptive geneticvariation that can be used in any organism (McDonald 1994, Kreitman 2000, Yang and Bielawski 2000, Beaumont and Balding 2004, Beaumont 2005). When these tools can be applied to organisms whose ecology is both well understood and readily manipulated, deeper insight into the biological signiﬁcance ofgenetic polymorphisms can be obtained. Such a synthesis is a goal of this paper.
The authors suggested that lack of connection between up- and downstream populations due to damming led to population fragmentation of S. hilarii in the area studied. Similarly, we found strong genetic differentiation (for mtDNA, F ST = 0.109; for microsatellites, D EST = 0.1269) between the Igarapava and Lavras populations on the rio Grande, separated from each other by 422 river km and four hydroelectric dams. The genetic differentiation we observed among S. hilarii populations in the rio Grande may be a combination of historical differentiation and recent effects of gene-flow disruption caused by the dams followed by reproduction of isolated spawning assemblages in mid-sized tributaries of the respective reservoirs. The contribution of contemporary lack of gene flow to historical differentiation is difficult to assess, although we note that the mean value for the mitochondrial F ST value (0.192) was slightly greater than that for the corresponding nuclear metric, D EST (0.181). We suggest spatially more intensive sampling of S. hilarii populations across the rio Paraná system in order to more effectively distinguish between historical and contemporary differentiation. Our data set also provides the basis for future studies of temporal variation in geneticvariationof S. hilarii in the system. Similar studies of S. hilarii might be undertaken in other river systems, prioritizing those were dams have been or might be constructed.
We are interested in understanding the population genetic structure, patterns of gene flow and mating system of T. papyrus, to generate useful information for conservation strat- egies. As part of this project we report here the development of 12 microsatellite loci for the species.
Soil physical characteristics have been shown to be the main factors controlling spatial patterns of soil water and soil nutrients (Zhao et al., 2011). Soil compaction follow- ing heavy grazing can lead to a homogeneous spatial dis- tribution of soil characteristics and increase the vulnerabil- ity of soil water and soil loss, and consequently reduce wa- ter availability for plants and rangeland production (Zhao et al., 2011). With increasing grazing intensity, heterogeneity of soil and plant characteristics changed from a patchy to a homogeneous distribution (Zhao et al., 2011). This was also confirmed in our study where heavy grazing decreased vari- ation in soil properties (SWC, SOC, TN) at the 10 m scale, strongly modified soil property patterns and changed species composition. In our study, 4 years after grazing intensity was changed from heavy grazing to moderate grazing at the MG site, spatial variability of soil properties increased at the MG site compared to the HG site. Livestock grazing resulted in changes to litter input, which may have influenced SOC (Zhao et al., 2005; Su et al., 2006; Lin et al., 2010; Komac et al., 2014). Although Lin et al. (2010) thought those factors resulted in a more homogeneous grazing distribution, they were not strong enough to alter the pre-existing spatial pat- terns of vegetation and soil fertility in their desert steppe. They found that neither SOC nor total N responded to graz- ing intensity at a coarse scale (1–18 m), while soil water con- tent and SOC decreased with increasing grazing intensity at a finescale (< 2 m) (Lin et al., 2010). Our findings from the typical steppe confirmed that livestock grazing can change the spatial patterns of soil properties at a 10 m 2 scaleand make them more homogeneous. The heterogeneity of SOC patches decreased with increasing grazing pressure, which was in agreement with the long-term (> 25 years) responses of SOC spatial patterns to grazing in a semi-arid steppe in Inner Mongolia (Wiesmeier et al., 2009). This was also con- firmed by the findings of Augustine and Frank (2001) who found that semivariance was positively correlated with dis- tance between sampling points in grazed grassland at Yellow- stone National Park, indicating that homogeneous patches occurred at a scale > 30 m for soil N.
Ecological processes that determine the abundance of species within ecological com- munities vary across space and time. These scale-dependent processes are especially important when they affect key members of a community, such as ecosystem engi- neers that create shelter and food resources for other species. Yet, few studies have examined the suite of processes that shape the abundance of ecosystem engineers. Here, we evaluated the relative influence of temporal variation, local processes, and latitude on the abundance of an engineering insect—a rosette-galling midge, Rhopalomyia solidaginis (Diptera: Cecidomyiidae). Over a period of 3–5 years, we studied the density and size of galls across a suite of local experiments that manip- ulated geneticvariation, soil nutrient availability, and the removal of other insects from the host plant, Solidago altissima (tall goldenrod). We also surveyed gall density within a single growing season across a 2,300 km latitudinal transect of goldenrod populations in the eastern United States. At the local scale, we found that host-plant genotypic variation was the best predictor of rosette gall density and size within a single year. We found that the removal of other insect herbivores resulted in an increase in gall density and size. The amendment of soil nutrients for four years had no effect on gall density, but galls were smaller in carbon-added plots compared to control and nitrogen additions. Finally, we observed that gall density varied several fold across years. At the biogeographic scale, we observed that the density of rosette gallers peaked at mid-latitudes. Using meta-analytic approaches, we found that the effect size of time, followed by host-plant geneticvariationand latitude were the best predictors of gall density. Taken together, our study provides a unique comparison of multiple factors across different spatial and temporal scales that govern engineering insect herbivore density.
Allele and genotype frequencies were estimated for all loci. Hardy-Weinberg equilibrium (HWE) was tested using an exact test, based on a Markov chain approach. An unbiased estimate of heterozygosity was computed according to Nei (1978). The statistical significance of the variance com- ponents of the AMOVA (considering the island as the main group) and the paired comparisons were determined by non-parametric procedures. Pairwise population differen- tiation exact tests, using allele frequencies, were carried out to compare the data obtained for the three islands. Allele frequencies for the total sample (n ¼ 305) were compared with the available data for other European and non- European populations. Data from mainland Portugal were taken from David et al. (2004), Ferrer-Antunes et al. (1998), Magro et al. (2003), Medeiros et al. (2002), Mendonc¸a et al. (2008) and Rodrigues et al. (2005). For the remaining populations the data used in the differentiation tests were taken from the ALlele FREquency Database (http://alfred. med.yale.edu/alfred), SNPedia (http://www.snpedia.com), Entrez SNP database (http://www.ncbi.nlm.nih.gov/SNP). All population analyses were performed using Arlequin software version 3.0 (Excoffier et al. 2005).
Abstract: The development of fisheries sector is intended to improve the role of creating a strong linkage with other sectors by increasing the value added, absorbing labor forces and increasing people’s income so that this can make the economy grow well. The value added is a value that increases due to a commodity that has been processed, transported or stored in a production. Lamongan and Pelabuhanratu regencies are one of fisheries centers on the north and the south coast of Java Island. The aim of this research was to know the value added and the business margin of fisheries from the processing and marketing aspects. The research was carried out in two locations; Northern coast (Lamongan regencies) and Shouther coasts (Pelabuhanratu regencies), Indoneisa. The data used were primary data; the people involved in the business including fishing, marketing and processing product. The results showed that the process of fisheries product yielded the value added and margin that were created from the incorporation of business benefit, added input contribution/ other input and direct reward for the labor forces. The value added and the business margin of product processing can reach 2 to 3 fold from the main input value. The value added and the business margin of fisheries product processing were very big. This was the source of economy growth there. The effort to develop the business of fisheries product processing in the small scale need to be supported with various programs especially in the market access and funding.
Relational Databases had been introduced in 1970 by Edgar Codd at IBM Almaden Research Center [Cod70]. It has been used for decades and has brought in the concept of relations. With this, a relational database is usually represented by a structured model, a table, in which each row is a tuple (also considered as an object) and the columns are the attributes of the tuple. The database can be queried in order to retrieve useful data based on their attributes whether in common between tuples or not. To do so it is used a "structured query language", SQL [LM10]. The main set of operations is known as CRUD: Create, Read, Update and Delete. Relational databases are also know for their fixed schemas that force every tuple to have the exact same attributes.
The previous work was analyzed and criticized by Hellmann (1998b). The author defends that the venture capital is extremely important in the countries competitiveness, particularly in the case of the U.S.A, and argues that the lack of venture capital is essentially due to the lack of entrepreneurs. According to the author we still do not know the entrepreneurship process and thus there does not exist a correct form of measuring the entrepreneurship level. One of is recommendations is that instead of using the variables in absolute terms they should be used as fractions of the GDP or of the saving level. In our study we follow this recommendation as other authors have already done it. By doing this we obtain values which are normalized with respect to the different economic growth and different inflation rates. Finally, this author suggests that it would be interesting to verify the impact of the age of the venture capital company using an analysis with disaggregated data. He also suggests a deeper investigation of the early stage venture capital investments.
For each TU, all possible transcript combinations, or transcript pairs (TPs), were collected. To investigate whether two transcripts in a TP are alternatively spliced, an algorithm was developed. Basically, given a TP, we compared the exon coordinates of one member with those of the other member. An AS event was defined by the exon coordinates comparison status. There were three states of comparison between an exon and a transcript: falling outside the coordinates of the transcript, overlapping with an exon of the transcript, and completely falling inside an intronic region. Exons with coordinates that fell outside of the transcripts were not informative, thus we only considered the last two states in the following analyses. For an internal exon, an AS event was considered as either completely mapping within the intronic region or overlapping with the exon but extending or truncating at one or both ends. For a 59 or 39 terminal exon with overlapping status, only extending or truncating at the interior end was considered an AS event. For a 39 terminal exon, if it was mapped completely to the intronic region of another transcript, an AS event was considered. TPs with one or more AS events mentioned above were considered alternatively spliced and grouped as AS- TPs. There was another AS event, which was derived from a situation in which the 59 terminal exon was completely mapped to intronic region of another transcript. We treated this type of AS event separately, and TPs with only this AS event were classified into another TP group named C5T-TPs. The reason for this was that our intention was to assess the hypothesis that APs have regulatory effects on AS, whereas this type of AS event is definitely correlated with TSS, and thus not informative. TPs without any of the AS events were grouped as Non-AS-TPs (Figure 1).
Foraging strategies of marine predators have evolved to respond to patchy prey distribution in a vast, heterogeneous and dynamic environment. These strategies allow predators to locate biological hotspots, where the probability of prey encounters is higher, reducing travel speed and increasing their turning rates in order to adjust searching effort in response to variations in prey densities, the so-called area-restricted search (ARS) behaviour, and to the surrounding environment. Due to their unique oceanographic characteristics, oceanic fronts (horizontal gradients in water properties, such as temperature, salinity, among others) are highly productive environments and consequently, foraging hotspots for many marine species. However, despite fronts importance as foraging locations, their ecological value for top predators is not yet fully understood. Thus, using fine-scale composite front maps and tracking data from individuals tagged in the north Atlantic over 9 years (2006-2015), I aim to understand the influence of thermal front gradients (in oceanic fronts) on the foraging behaviour of blue (Prionace glauca) and mako (Isurus oxyrinchus) sharks. Specifically, I investigate the relation between sharks’ FPT as measure ofindividuals’ search effort, and front metrics (intensity, proximity and frequency) using Generalized Additive Mixed Models (GAMMs). According to the results, higher frontal intensity (F comp ) values lead to an increase in the
Extensively self-renewing erythroblasts (ESREs) were derived from E13.5 mouse embryonic fetal liver, as previously described , using STEMPRO34 medium. These cells are dependent on erythropoietin (EPO), stem cell factor (SCF), and dexamethasone for their ex-vivo self-renewal. Proliferating erythroblasts mature into erythrocytes when cultured in the absence of dexamethasone, in erythroid maturation media, which contains IMDM, EPO, SCF, serum replacement, PDS (plasma-derived serum), glutamine, PFHM-II (protein-free hybridoma media) and MTG (monothio- glycerol). Detailed protocol has recently been described . Cell size was measured using forward scatter parameter (FSC). ESRE cell differentiation was monitored using a FACSCanto II flow cytometer (BD Biosciences, Le Pont-De-Claix, France) after triple labeling with the following monoclonal conjugated antibodies: anti-CD71–fluorescein isothiocyanate (FITC), anti-Ter119–phy- coerythrin (PE) and anti-CD117-allophycocyanine (APC). All these antibodies were purchased from Caltag Laboratories (Burlingame, CA). Cells were collected for protein and RNA analyses.