CRM Prediction and CRM Validation Approaches

CRM Prediction and CRM Validation Approaches Since CRM is underlying the regulation of gene expression in tissue-specific manner, understanding the characteristics of CRMs is helpful to determine the potential CRM candidates for further applications such as tissue-specific gene therapy. As previously discussed the influential parameters to CRM activity include the types and arrangement of transcription factor binding sites (TFBSs) and epigenetic modification pattern[121, 124]. Therefore, these factors are taken into account for prediction of promising CRMs. Transcription factor binding sites are described as short DNA regions (6 to 10 bp in length) which are recognized and bound by various transcription factors[149]. One CRM can contain many TFBSs depended on its functionality[150]. Several experimental studies have been performed in order to map the TFBSs in DNA genome. Chromatin immunoprecipitation (ChIP) assay is a common method to identify the TFBSs in protein-bound DNA complexes in the solution[151, 152]. In addition, DNase footprinting, which relies on the digestion of exposed DNA region where it is not protected by target proteins, has also been used[153, 154]. The difference between these techniques is mainly involving resolution of transcription factor binding sites[155, 156]. To derive the TFBS motifs from raw data, these DNA sequences are used as the input to compute the similarity and the potential motifs are generated. To apply the information of transcription factor binding sites motifs on CRM prediction, it is relatively simple as this method requires solely genomic DNA sequences. The predicted motifs are mapped to the original genome and prospective CRMs containing clusters of TFBSs are identified[124, 157]. Due to the enormous spread of motifs in large genome, a lot of DNA regions showing the potency of being CRMs are indicated; however, only few DNA sequences are actually occupied by the target transcription factors[158]. In the erythroid cells of mouse genome showed approximately 8 million hits of GATA-binding factor1 (GATA1) binding site motifs, but only 15360 motifs were bound by GATA1 and all of bound motifs bore H3K4 monomethylation[159]. Indeed, relying on merely TFBS motifs is not sufficient to obtain the significant CRMs. The study on smaller-size genomes is one alternative to improve the quality of CRM prediction.[157] Another approach to determine the potential CRMs is the use of conservation of non-coding DNA among several species. The assumption is that the DNA sequences associate with gene expression are highly conserved in comparison to non-essential DNA after evolving through the purifying selection over time [157]. This method is not depended on the information on TFBS so that it offers another solution to prediction of CRMs in case tissue-specific enhancers have not been widely studied. At initial study about the DNA sequence alignment of more than 100 bp-long DNA between human and mouse, with the minimal conservation of 70%, was conducted and potential enhancers for certain genes such as interleukin-4, interleukin-13 and interleukin-5 were identified[160]. Later on this approach shows the promising results due to high validation rates in transgenic mouse embryo by using rigorous conservation constrain[160-163]. The conservation-based prediction is also applicable to discover novel TFBSs wh ere the information is not extensively elaborated. With the DNA sequence alignment between orthologous species, the short DNA sequences conserved in many species, namely phylogenetic footprints, could be the possible binding sites for transcription factors [164, 165], and mutations of the conserved boxes can lead to the reduction of gene expression as in the example of altered effect of variant E box on ÃŽ µ-globin reporter gene induction[166]. As the approach is mainly related to the evolutionary constrain among species it means that the use of this method may overlook the potential CRMs which are lately developed and the TFBS pattern cannot be aligned to the former population[157]. For example, in the ChIP-seq study the GHP68 enhancer, located at intragenic region of mouse abhydrolase domain containing2 (Abhd2) gene, does not contain the footprint of GATA-binding factor1 (GATA1) motif which is commonly found in Abhd2 genes of other non-primate species[167]. Indeed, the GHP68 enhan cer in primate genome possesses the unique protein binding pattern[157]. Another consideration on conservation-based prediction is that even though the conservation level of selected CRMs is extremely high among orthologous species, the actual activities of CRMs possibly vary from species to species in nature[168]. Due to the limitations of previous approaches regarding false positive prediction by highly redundant presence of TFBS motifs in large genome[158], as well as lineage-specific evolution of certain CRMs in different organisms[157], epigenetic regulation is considered the promising parameter of CRM prediction as a result of the strong correlation between hypersensitivity to DNA treatment/histone modification and enhancer activity[169-171]. Many CRMs have been found to localize at genome region where the response to DNase activity is very sensitive[153, 172]. In addition biochemical patterns of modification at enhancer are showed including histone acetylation[169], high H3K4me1 as well as low H3K4me3 modification[170], and occupancy of histone acetyltransferase p300[171, 173]. For active promoter, in contrast to usual enhancers, the major characteristic is the presence of nucleosome-free and high level of H3K3me3 modification[174, 175]. By using the reference genome database containing epigenetic as well as DNase hypersensitivity regions, where the information is obtained from ChIP seq [176], and DNase seq experiments, the substantial rate of validation of selected CRMs from 43 to 100% in many study models[169-171, 176, 177] indicates the robustness of the epigenetic-based approach. The idea is this method is optimized that the predicted conditions is not too stringent as evolutionary conservation method and the number of output is not too enormous as TFBS-based prediction[157]. Still, some potential CRMs can be overlooked using biochemical features[173, 178]. For instance, the study of heart enhancer identification showed that three different predictions yielded various amount of outputs. The possible CRMs were hardly obtained through comparative genomic DNA alignment while the use of p300 occupancy to identify the potential sequences gave rise to 130 output sequences with 75% validation rate[173]. In another TFBS-based study in heart by Narlikar and colleagues, the classifier, where its database relied on predicted and validated TFBS, was generated to select the putative CRMs from the non-functional DNA[178]. This prediction allowed them to distinguish 40,000 CRMs from genome and the validation rate was relatively considerable in comparison to the epigenetic approach[178]. This suggests the need of additional further study on biochemical pattern prediction to cover the missing CRMs. Using experimental and computational study, scientists are able to collect the extensive information about TFBSs, epigenetic modification and conservation of DNA among species. This data has been widely deposited in many open-access database websites, which become the significant information resources for further CRM identification[179]. The Ensembl Regulatory Build is recently developed to integrate the previous discovery of epigenetic marks and occupancy of transcription factors from different projects and build the better-defined regulatory regions in human genome[180]. Another commonly used database website is the University of California Santa Cruz (UCSC) Genome Browser Database, which provide all aspects of information for CRM prediction including experimental (DNase hypersensitivity clusters, epigenetic marks of histone proteins, and binding of transcription factors from ChIP seq) as well as computational (conservation level among vertebrates from DNA sequence alignment) study [181]. This aids the feasibility of enhancer prediction since the use combinatorial information would suggest more significant CRM outputs with higher validation rate[182-184]. For example, the sophisticated protocol designed by Nair and team to identify the liver-specific CRM was derived from the integration of experimental study from UCSC genome browser and the putative TFBS motifs from computational analysis[182]. To obtain predicted liver-specific TFBS motifs, the presumptive promoters, which are 1000-bp DNA sequences located upstream of transcription start sites, from highly-expressed genes were initially compared to ones from low-expressed genes in the liver, followed by computing the potential TFBS motifs which are likely to associate with liver-targeted gene induction based on distance difference matrix (DDM) and multidimensional scaling (MDS)[182, 185]. The DDM was primarily used to identify the difference between two protein structures by calculating the distance differenc e values from low distance matrices[186]. Ultimately the predicted TFBS motifs were mapped to the corresponding DNA sequences of liver-specific genes in UCSC genome browser where the experimental data of such genes was previously described[182]. The ideal CRMs were expected to show the coexistence of predicted motifs together with dense DNase clusters, high conservation level in vertebrates, and explicit histone modification patterns. In addition, the putative motifs should be consistent to the transcription factor lists from ChIP-seq experiment. The promising liver-specific transcriptional module from prediction was further validated and showed the remarkable activity to up-regulate hFIX expression up to 15 fold compared to control, reflecting the robustness of the prediction method[182]. The same approach has also been applied to design the CRMs targeting other target cells such as cardiomyocytes, and the 10-fold augmented expression of cardiac genes was noted upon validation in m ouse model[183]. Taken together, this suggests the increased power of using multiple parameters to determine transcriptional modules, and the combined data provided in UCSC genome browser is valid; the integrated data is nicely standardized so that the summary of information is reliable. However, the feasibility of combinatorial approach, relying on both computational data and previous experimental study, is the major concern due to the requirement of strong expertise on bioinformatics knowledge for computation of TFBS motifs. One possible alternative to circumvent this limitation would be the direct use of available information on UCSC Genome Browser for CRM selection by taking associated determinants (DNase hypersensitivity, transcription factor binding, histone modification, and conservation level among vertebrate) into consideration. There are several validation assays that have been performed to investigate the potency of CRMs to enhance gene expression. In general, the plasmids containing minimal core promoters and reporter genes such as lacZ, encoding ÃŽ ²-galactosidase, luciferase, and green fluorescence protein (GFP), are the backbone constructs, and the predicted CRM are cloned into certain position based on the validation methods[149]. Usually CRM sequences are inserted at the upstream of the promoters and the increased strength of overall construct expression is assessed after transfection or integration of plasmids[187-196]. In order to develop the downstream process to identify the target cells where CRMs are active, the use of heterologous barcode has been done so that the number of CRM high-throughput screening is up to hundreds or thousands [191-194, 196]. In some studies, the need of barcode is eliminated by targeting at enhancers directly, and the method is called self-transcribing active regulator y region sequencing (STARR-seq) [197]. Both transgenic animal embryos and specific cell lines [187-191, 193-196] are commonly used to study CRM activity. For example, transgenic mouse or fly (D.melanogaster) containing putative CRMs as well as reporter genes are initially generated, and the development of reporter gene signals later observed at the certain parts of embryos is identified depended on tissue specificity of CRMs[198]. To improve time and cost-effectiveness of the current approach, Gisselbrecht and colleagues developed the technique called enhancer-FACS-Seq (eFS), which makes use of the distribution of GFP signaling based on the tissue-specific CRM enhancement, to sort out the GFP-positive cells from the negative population using fluorescence activated cell sorting (FACS)[190]. Validation of the effect of CRMs on gene expression has also been reported in animal models and the delivery methods of CRMs are adjusted to be tissue-specific. AAV is the example of tissue-target ed delivery system since its tropism is relied on the serotype[182-184]. The use of AAV vectors to carry the predicted CRMs to the specific organs has been done in heart and liver enhancers by using AAV9, and the follow-up process was achieved through the reporter hFIX protein expression in the blood. In murine models, to reduce the cost of virus production, HD injection of plasmids containing CRMs in mice can be primarily done for initial screening[182]. This method is distinctive since the model simulates the actual situation of CRM activity in animal body for gene therapy application[182-184]. In addition, another advantage of using this approach is the longevity and the expression level can be observed continuously for long-term study as the mouse sacrifice is not required. Biology of hepatocellular carcinoma (HCC) Hepatocellular carcinoma (HCC) is one type of liver cancers which is highly prevalent in many regions such as East Asia, Africa, and United State[199]. Even though the incidence of HCC ranks the sixth in comparison to other cancers the rate of mortality is relatively high[200]. There are several etiological factors describing HCC development including Hepatitis B (HBV) and C (HBC) infection, aflatoxin-directed induction, alcohol consumption, accumulation of fat in the liver resulting in non-alcoholic steatohepatitis (NASH), sex-related influence, unbalance of microbes in gastrointestinal tract, and type II diabetes[201]. Each factor has specific mechanism to cause HCC, but in general most of factors ultimately lead to liver cirrhosis formation and subsequently HCC[202]. A number of staging system to classify HCC disease development stage have been designed for diagnosis; however, the gold-standard for staging remains challenging due to heterogeneity of HCC population[203]. To study the molecular mechanism underlying HCC development, copy number genomic[204-206], exomic[207, 208], whole-genome sequencing[209, 210], and transcriptomic[211, 212] studies have been conducted in liver cancer tissues. In copy number alteration analysis, both deletion (i.e. TNFAIP3, CDKN2C, WRN, PTEN, BRCA2) and duplication (MDM4, BCL9, ARNT, MET) of specific genes are found in HCC genomes[213]. Exome and whole-genome sequencing in HCC allow detailed investigation of genome structures at the levels of mutation in both coding and non-coding regions[213, 214]. For example, mutation of NFE2L2-KEAP1 and MLL genes were identified from 87 cases with HCC development using exomic approach[214]. Transcriptomic study gives another insight into HCC regarding the change of expression profiling compared to normal hepatocytes. Using in combination with whole-genome sequencing, transcriptome revealed the RNA editing mechanism implicating in up-regulation of gene expression in cancer developm ent[215, 216]. Taken together, the aberrant genes found in HCC are mapped to cellular pathways to explain the molecular mechanisms underlying disease development. The pathways which are postulated as the keys for hepatocarcinogenesis include cell cycle regulation (i.e RB[217], CDKN2A[218]), WNT pathway (i.e. APC[219], AXIN1[220, 221]), chromatin remodeling (i.e. ARID2[208, 210], MLL[222]), tyrosine kinase signaling (i.e. SOCS-1[223], IGF[224]), and NOTCH[225, 226] pathways. Apart from structural genes, miRNAs, small non-coding RNAs which control gene expression at post-transcriptional level through hybridization with the mRNA templates and subsequently leading to translation inhibition or RNA degradation[227], are implicated in HCC progression due to the evidences on differential miRNA expression between HCC and normal hepatocytes[228, 229]. In general, miR-92, miR-18 and miR-20 are significant in HCC stage progression[229]. Some altered miRNA expression is associated with etiological factors. For[MC1] instance, there is correlation between miR-126 down regulation and alcohol consumption[230]. The functions of miRNA in HCC pathogenesis are divided into two groups; oncogenic miRNAs and tumor-suppressor miRNAs. For oncogenenic miRNAs, three miRNAs including miR-221, miR-224 and miR-21 have been showed to enhance hepatocarcinogenesis. The miR-221 plays role in cancer invasion using two mechanisms; increasing cell proliferation targeting CDKN1B/p27 expressi on[231], and enhancing cell migration through AKT signaling[232]. The invasion of HCC is also supported by miR-224, but its mechanism of action is involved with homeobox D10 downregulation and induction of inflammatory pathway[233]. Another oncogenic miRNA miR-21 is reported to suppress expression of program cell death 4 (PCD4) [234, 235]protein which functions as tumor suppressor protein, and to increases cell proliferation through the regulation of mitogen-activated protein kinase-kinase 3 (MAP2K3) activity[236]. Apart from individual miRNAs, certain clusters of miRNA have been identified to contribute to HCC progression. For instance, the up-regulation of miR-17-92 cluster, which is composed of miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1[237], was found in HCC, and the attenuation of its expression diminished the ability of malignancy transformation[238]. The activity of miR-17-92 cluster affects the expressions of certain genes usually found in HCC such as PTEN, E2F1, and E-cadherin[239]. However, the individual miRNA members may function in the different ways. For example, up-regulation of miR-19 suppressed the formation of liver fibrogenesis through TFF-ÃŽ ² signaling[240]. A number of tumor suppressive miRNAs have also been discovered to diminish HCC development. The miR-122 function is to control the genes associated with tumor formation and metastasis including VEGF[241], RHOA[241], PKM[242] whereas miR-375 exerts its activity by suppression of ATG7 expression to block autophagy[243], the essential mechanism of cancerous cells to survive under hypoxic environment. The miR-125b prevents cancer proliferation by activation of p21(WAF1/Cip1) G1/S cell cycle arrest as well as repression of SIRT7 gene induction[244]. G1/S transition of cancer cells is also controlled by miR-26a activity[235]. The overall functions of HCC-associated miRNAs are implicated in STAT3, by modulating Bcl-2 and Mcl-1 functions, and NF-ÃŽ ºB inflammatory pathways, le ading to hepatocacinogenesis[245].

Expository Essay – Cory and Noynoy Aquino

Aquinos: Cory and Noynoy on Politics Cory and Noynoy were both known in the field of politics; they have their hopes, visions and actions toward change to help our country – in the area of economic development in general, and as well as its citizens. They also had several major accomplishments that benefited the Filipinos and helped the nation to cope up from despair. They both had their start in the said realm. Cory being the first one to step on the governance than Noynoy, started in year 1986 and noted as the first female president of the Republic of the Philippines and Asia.Noynoy, following the examples left by his gallant parents took part in politics and considered as the fourth-generation politician of their family; He entered in year 1998 as a member of the House of Representatives from Tarlac's 2nd district and now as the 15th and current president of our nation (since June 2010). Both of them envisioned and took actions to provide a better tomorrow to every Filipino . Cory’s motivation in handling governance was to bring back economic health and confidence after Marcos’ abusive acts under his reign that brought misery to our country.She also envisioned and made achievements that; First, agrarian and land reform as the centerpiece of her administration's social legislative agenda. Second, she reinstated the writ of habeas corpus, the right of a prisoner to appear before a judge, and abolished the government's ability to imprison people at will, which had been in effect since 1981. Third, she promised to promote the right to assemble peaceably, and free speech along with prosecuting corruption and abusers of human rights.Fourth, she said she would revitalize the sugar industry by breaking the monopoly. She acknowledged the special relationship with the United States but emphasized that her concern was with the Filipinos, not the Americans. Even after her sovereignty, she was heavily involved in several charitable activities and soci o-economic initiatives. She supported other causes such as the Gawad Kalinga social housing project for the poor and homeless. Noynoy on the other hand, continuously pushes for legislation which helps Filipino workers and consumers. The President also nvisioned and takes actions that: First, in 2016 tourist arrivals will figure at 10 million; Second, by next year he forecasted that we will be a rice sufficient country and will have the capability to get back as a rice exporting country; Third, infrastructure projects for the next year; Fourth, strengthening our defense capabilities especially now that we are in dispute with China over the West Philippine Sea; Fifth, his call for the congress to amend the Anti-Money Laundering Act, the revision of the Mining Law which will increase the percentage of the government’s share in the revenues from mining; Sixth, the passing of the sin tax law and the government’s achievement in the fight against corruption through the succes sful impeachment trial of the former Chief Justice Renato Corona, and many other countless plans for the Philippines. Cory and Noynoy achieved major accomplishments during their administration. First, on Cory’s term, she restored Philippine democracy and provided freedom for the citizens.Second, she improved agrarian and land reform; And after stepping down from the presidency, Cory Aquino remained active in helping nurture the fragile Philippine democracy. Largely through the Benigno S. Aquino, Jr. Foundation (BSAF), she supported programs that promoted people empowerment, peace and human rights. In the final years of her life, she devoted much time and energy to harnessing private sector support for the microfinance sector. Her vision was to strengthen the infrastructure that would transform micro-enterprise development into a potent vehicle for raising â€Å"people power† to the next level, slowly creating a broad middle class that would fortify the foundations of P hilippine democracy. Meanwhile, on Noynoy’s period of influence, the country had gains in tourism and agriculture.He also continuously works on his pursuance for a corruption-free country and other numerous concerns that our nation faces. President Cory did not pursue for more time in power after her term. She strongly declined the requests for her to seek reelection for she wanted to set an example to both citizens and politicians that the presidency is not a lifetime position. Cory and Noynoy are both effective leaders; They may have different advocacies but their goal centers on change and a brighter future for our country. They both started on politics, had visions and pursues for change as well as success and had carried out major accomplishments for the Filipinos.

The Basics of 50 Essay Topics That You Can Learn From Beginning Right Away

The Basics of 50 Essay Topics That You Can Learn From Beginning Right Away The readers should realize that you have in-depth understanding of the area. The absolute most famous American writer who influenced the entire nation. Whether you currently have a topic in mind or want guidance in locating the very best option possible, our team members will be able to help you create a 100% unique and authentic essay that will satisfy all your academic requirements. Sports research paper topics cover a wide variety of fields. Introducing 50 Essay Topics Overall, a narrow topic will supply you with clues about what kind of examples you should put in your essay so you ought to strive for narrow instead of broad topics. In many professional contexts, respectful argumentation is the thing that leads to the growth of new thoughts and perspectives. When it has to do with writing an argumentative essay, the most essential matter to do is to select a topic and an argument you may really get behind. The very first point to do is to make an outline of the topic you pick. Starting off big can also provide your readers plenty of big expectations from your composition. The essay is composed of your own personal views on the topic and an explanation of your position. Whether you're looking for good narrative essay topics or aren't certain how to choose the most appropriate one from the list of good descriptive essay topics, make sure to reassess our topics' selection as we guarantee you will definitely find something to fulfill your requirements. Anyway, a topic for synthesis essay shouldn't be too broad so you could develop a suitable argument. It's important to select debatable argumentative essay topics since you need opposing points you can counter to your own points. The Key to Successful 50 Essay Topics Many brilliant individuals who achieved success in life proved actually academic drop-outs. Pay close attention to all things electronic, and you will be certain to find something debatable of what you see. When you're picking your topic, bear in mind that it's much simpler to write about something which you presently have interest ineven in case you don't know a great deal about it. Explore the beneficial and negative impacts of this shift. The Downside Risk of 50 Essay Topics Despite the fact that you pay for homework, we provide those options at no cost. If you see that you're unaware of any topic, the easiest was to manage the undertaking is to get a high-quality customized essay online at Essay-Online-Shop. Imagine you own a time machine. When choosing a topic, there is not any systematic method to approach it. Essay Topics Secrets Exemplification essays enable students to obtain a deeper knowledge of theories, together with enable students to write with more skill, all while having the ability to internalize concepts wholly. Students lead busy lives and frequently forget about an approaching deadline. Learning a new language for an early age is helpful for kids. Don't neglect to write together with your students. 50 Essay Topics for Dummies Moral argumentative essay topics are a few of the simplest to get carried away with. If making your essay creative, you will unquestionably get the maximum grade. Argumentative essays are frequently more philosophical and synthetic essays are devoted to a specific matter. Basically, an exemplification essay is a kind of argumentative essay. Essays usually ask that you explore recent socio-political-economic developments. Researching the topic will permit you to find out more about what fascinates you, and should you pick something you really like, writing the essay will be more enjoyable. Argumentative essay topics are so important since they are debatableand it's critical to at all times be critically contemplating the world around us. As soon as you're prepared to think of a thesis, take a look at these Argumentative Thesis Statement Examples. If you're looking for descriptive essay examples here's a great one below. You'll have about 15 minutes to compose your essay.

The Holy Of The Worlds And Allah Akbar - 1604 Words

Islam Park University Desmond Hutchinson LE300 All Praise is due to Allah, the Lord of the Worlds and Allah Akbar Phrases that maybe more familiar to western culture in current times (Supreme wisdom 2012). Where do these statements originate from? Where do these beliefs come from? The people of Islam hold many core beliefs within their religion with the upmost importance; they utilize their interpretation of the Qur’an and the teachings of the Prophet Muhammad as a way to shape their lives. Origin of Islam Islam initially emerged from Mecca in Arabia during the seventh century. The well established city of Mecca and its surrounding area of vast deserts barren hills and valleys were home to the Inhabitants of the Bedouin Tribe†¦show more content†¦Muslims like most other religions are monotheists, they believe in only one god. Islam originated in some of the surrounding areas as other religions such as Christianity and Judaism. Muhammad at the age of 40 had a divine experience in which Muslims believe that the angel Gabriel spoke to him, speaking the actual words of god himself. Muhammad was told by the angel Gabriel that he had been chosen as a prophet by god and that he must repeat the words that he would be given to all of those who would listen. (Hemeyer, 2010) Islam first began as an oral tradition from Muhammad to his close associates and then to the world. The Prophet Muhammad was just and man, a human just like all other people. Muhammad is held in incom parable greatness and the basis of what an ideal man should be like in the Islamic faith. The stories of his life are held as an inspiration for the followers of Islam. His words and actions were recorded by his followers and these recordings where the basis for the holy doctrine of Islam in the Qur’an. The interpretation of the Qur’an is applied to the situations of life for the people of Islam. The love and devotion for Muhammad can be found in every language spoken by Muslims; standards of living are not only taken from the Qur’an but from the life and stories of Muhammad as well. The Qur’an or the Holy Qur’an is the sacred scripture that was originally