post-Human Genome Project Era Research Articles

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

Catching global interactions in vivo

Histone proteins and transcription factors (TFs) play critical roles in gene transcription and development of multicellular organisms. Although antibody mediated protein isolation couple with mass spectrometry approach has been a standard method to identify TF interacting partners and characterize their functional molecular complexes, it becomes urge to develop a robust method to functional characterize how these transcription factors act during biological process in the post-human genome project era. Here, Dr. Zhao and his colleagues in the National Heart, Lung, and Blood Institute of NIH develop a sensitive and robust strategy to globally identify and characterize in vivo protein–protein interactions termed bait protein–protein interaction-sequencing (bPPI-seq) (Zhang et al. in Cell Res doi:10.1038/cr.2017.112, 2017). As a proof-of-principle, they demonstrated that genome-wide interacting partners of histone variant H2A.Z are mainly involved in transcriptional regulation which is distinct from the interacting proteins of canonical histone H2A. Thus, their results suggest that bPPI-seq can be widely used to globally characterize protein complexes especially transcription factor interacting partners and molecular networks formed.

Plasticity and Programming: Feminism and the Epigenetic Imaginary

AbstractThe new science of epigenetics has raised hopes for an embrace of greater plasticity and variation within the biology of sex, gender, and sexuality than previously appreciated. This essay describes and analyzes the integration of epigenetics research into the scientific study of core biological pathways related to sex, gender, and sexuality in the brain in the post–Human Genome Project era. Through a close reading of the primary scientific literature, it demonstrates that epigenetic approaches in this subfield remain continuous with historically entrenched models of hardwired sexual dimorphism in the brain. Considering the opportunities and dilemmas of feminist engagements with the fast-moving and still-nascent field of epigenetics, it argues that for epigenetics to become a resource for studies of the development and plasticity of gendered-sexed bodies and identities, feminists must contest the ontological and epistemological commitments of mainstream research in this field. Feminist attraction t...

Construction of a versatile expression library for all human single-pass transmembrane proteins for receptor pairings by high throughput screening

Interactions between protein ligands and receptors play crucial roles in cell-cell signalling. Most of the human cell surface receptors have been identified in the post-Human Genome Project era but many of their corresponding ligands remain unknown. To facilitate the pairing of orphan receptors, 2762 sequences encoding all human single-pass transmembrane proteins were selected for inclusion into a mammalian-cell expression library. This expression library, consisting of all the individual extracellular domains (ECDs), was constructed as a Fab fusion for each protein. In this format, individual ECD can be produced as a soluble protein or displayed on cell surface, depending on the applied heavy-chain Fab configuration. The unique design of the Fab fusion concept used in the library led to not only superior success rate of protein production, but also versatile applications in various high-throughput screening paradigms including protein-protein binding assays as well as cell binding assays, which were not possible for any other existing expression libraries. The protein library was screened against human coagulation factor VIIa (FVIIa), an approved therapeutic for the treatment of hemophilia, for binding partners by AlphaScreen and ForteBio assays. Two previously known physiological ligands of FVIIa, tissue factor (TF) and endothelial protein C receptor (EPCR) were identified by both assays. The cell surface displayed library was screened against V-domain Ig suppressor of T-cell activation (VISTA), an important immune-checkpoint regulator. Immunoglobulin superfamily member 11 (IgSF11), a potential target for cancer immunotherapy, was identified as a new and previously undescribed binding partner for VISTA. The specificity of the binding was confirmed and validated by both fluorescence-activated cell sorting (FACS) and surface plasmon resonance (SPR) assays in different experimental setups.

Unravelling the genetics of inherited retinal dystrophies: Past, present and future.

The identification of the genes underlying monogenic diseases has been of interest to clinicians and scientists for many years. Using inherited retinal dystrophies as an example of monogenic disease we describe the history of molecular genetic techniques that have been pivotal in the discovery of disease causing genes. The methods that were developed in the 1970's and 80's are still in use today but have been refined and improved. These techniques enabled the concept of the Human Genome Project to be envisaged and ultimately realised. When the successful conclusion of the project was announced in 2003 many new tools and, as importantly, many collaborations had been developed that facilitated a rapid identification of disease genes. In the post-human genome project era advances in computing power and the clever use of the properties of DNA replication has allowed the development of next-generation sequencing technologies. These methods have revolutionised the identification of disease genes because for the first time there is no need to define the position of the gene in the genome. The use of next generation sequencing in a diagnostic setting has allowed many more patients with an inherited retinal dystrophy to obtain a molecular diagnosis for their disease. The identification of novel genes that have a role in the development or maintenance of retinal function is opening up avenues of research which will lead to the development of new pharmacological and gene therapy approaches. Neither of which can be used unless the defective gene and protein is known. The continued development of sequencing technologies also holds great promise for the advent of truly personalised medicine.

Mechanism of Label-Free DNA Detection Using the Floating Electrode on Pentacene Thin Film Transistor

The analysis of DNA in the posthuman genome project era has become an ever-expanding branch of research and is thus routinely employed in the majority of biochemical laboratories. This work discusses the mechanism and label-free detection of DNA using a pentacene thin film transistor with a gold floating electrode on the active layer. Thiolated polynucleotide probes were used, which form self-aligned monolayers on the floating electrode over the pentacene active layer. The immobilization of the DNA on floating electrode increased the work function and raised the Schottky barrier of the device, resulting in a charge screening effect. Hence, the negative charge of the DNA caused a positive shift in the threshold voltage of the transistor. Based on the change in the electrical output, synthesized DNA and the viral DNA were detected.

The genetics of human migrations

When our human ancestors began to migrate from the African savannahs across the Alps into Northern Europe and Asia between 200,000 and 60,000 years ago, they encountered a colder climate, different food sources and new predators. Inevitably, evolution began to work on those wandering tribes: their skin became lighter, their metabolism adapted to new food sources, and their immune system had to handle different pathogens. Modern Homo sapiens began to evolve into slightly different subspecies or, as they are referred to in the common vernacular, different races. This shallow notion of race—that differences in our appearance have deep‐seated consequences for who and what we are as human beings—is the root of racism, which has led to slavery, discrimination, murder and genocide throughout the millennia. With the emergence of modern genetics and genomics, scientists had hoped that a better understanding of genetic differences and similarities among humans would eventually help to overcome the controversial and toxic notion of race as having anything to do with a person's ability, intelligence or humanity. As J. Craig Venter, then head of the Celera Genomics Corporation in Rockville, MD, USA, put it in 2000, when the first draft of the human genome was published: “Race is a social concept, not a scientific one”. Since the publication of the human genome, further advances in genomic sequencing and statistical analysis are now yielding new insights into the link between past human migrations and today's variety of genetic differences that govern disease resistance, skin pigmentation, tolerance to cold, and ability to digest or metabolize various nutrients. Such findings are contributing to discussions around the delicate issues of race, ethnicity and identity, but they are also having a significant impact on biomedical research and personalized health care. Furthermore, genetic analysis is shedding light on the migrations themselves, along with …

Transcriptomics: Better Resolutions

Copyright: © 2014 Gupta S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In the post Human Genome Project era there are efforts to understand translation of the genomic sequence into the transcriptome. The human transcriptome is represented by >100,000 distinct transcripts presently described for ~20,000 protein-coding genes. Additionally, mRNA isoforms are produced by alternative processing of primary RNA transcripts. This alternative splicing affects >90% of the human genes and has been suggested to the primary driver of phenotypic complexity [1]. Despite this diversity in the coding sequence, the nonprotein-coding molecules contribute to > 95% of the transcriptome [2]. Numerous technologies and experimental platforms have facilitated the investigation of the complexity in the transcriptome that greater than that of the simple genome sequence.

MCU based real-time temperature control system for universal microfluidic PCR chip

The analysis of genetic materials in the post-human genome project era has become an ever-expanding branch of research and thus routinely employed in majority of biochemical laboratories. Most of the diagnostic research area emphasizes on polymerase chain reaction for detecting pathogenic organisms. However, the conventional polymerase chain reaction requires expensive and sophisticated thermal cycler and is not handy owing to its large dimensions. Therefore, we fabricated a continuous-flow polymerase chain reaction chip on a PDMS based microfluidic platform to ease the hardship of the conventional system. Temperature being the most crucial factor in polymerase chain reaction, was monitored and regulated by thermostatic action using an on-line computer system. Indium tin oxide coated glass platform was used for heating as it is transparent and has good thermal conductivity under the influence of DC bias. The heating circuit used an ATMega 128 MCU to control the temperature. As a result, a precise and quick heating environment was maintained on the microfluidic chip to amplify the target DNA. We successfully amplified Lambda phage and Escherichia coli DNA on our chip to prove the practicality of the device.

Translating the brain transcriptome in neuroAIDS: from non-human primates to humans.

In the post-human genome project era, high throughput techniques to detect and computational algorithms to analyze differentially expressed genes have proven to be powerful tools for studying pathogenesis of neuroAIDS. Concurrently, discovery of non-coding RNAs and their role in development and disease has underscored the importance of examining the entire transcriptome instead of protein coding genes alone. Herein, we review the documented changes in brain RNA expression profiles in the non-human primate model of neuroAIDS (SIV infected monkeys) and compare the findings to those resulting from studies in post-mortem human samples of neuroAIDS. Differential expression of mRNAs involved in inflammation and immune response are a common finding in both monkey and human samples - even in HIV infected people on combination antiretroviral therapy, a shared set of genes is upregulated in the brains of both infected monkeys and humans: B2M, IFI44, IFIT3, MX1, STAT1. Additionally, alterations in ion channel encoding genes have been observed in the human studies. Brain miRNA profiling has also been performed, and up-regulation of two miRNAs originating from the same transcript, miR-142-3p and miR-142-5p, is common to human and monkey neuroAIDS studies. With increases in knowledge about the genome and advances in technology, unraveling alterations in the transcriptome in the SIV/monkey model will continue to enrich our knowledge about the effects of HIV on the brain.

Capillary electrophoresis microchip for direct amperometric detection of DNA fragments

Detection and quantitation of nucleic acids have gained much importance in the last couple of decades, especially in the post-human genome project era. Such processes are tedious, time consuming and require expensive reagents and equipment. Therefore, in the present study, we demonstrated a simple process for the separation and analysis of small DNA fragments using capillary electrophoretic amperometric detection on an inexpensive disposable glass microchip. The device used polydimethylsiloxane engraved microchannel and Au/Ti in-channel microelectrodes for sample detection. The DNA fragments were separated under low electric field (20 V/cm) for improved detection sensitivity and to retain the biomolecules in their native conformation. With a low sample requirement (as low as 1 μL) and high reproducibility, the proposed microchip device was successful in resolution and detection of DNA fragments of various lengths.

Ancestry informative markers and parameters of complete blood count in Brazilian blood donors

In the post-Human Genome Project era, the debate onthe concept of race/ethnicity and its implications forbiomedical research are dependent on two critical issues:whether and how to classify individuals and whetherbiological factors play a role in health disparities. The adventof reliable estimates of genetic (or biogeographic) ancestryhas provided this debate with a quantitative and moreobjective tool. New techniques such as admixture mappingcan detect population-specific risk alleles for a disease inadmixed populations. However, researchers have to bemindful of the correlation between genetic ancestry andsocioeconomic and environmental factors that could underliethese differences.

Recent advances of genetic ancestry testing in biomedical research and direct to consumer testing

In the post-Human Genome Project era, the debate on the concept of race/ethnicity and its implications for biomedical research are dependent on two critical issues: whether and how to classify individuals and whether biological factors play a role in health disparities. The advent of reliable estimates of genetic (or biogeographic) ancestry has provided this debate with a quantitative and more objective tool. The estimation of genetic ancestry allows investigators to control for population stratification in association studies and helps to detect biological causation behind population-specific differences in disease and drug response. New techniques such as admixture mapping can specifically detect population-specific risk alleles for a disease in admixed populations. However, researchers have to be mindful of the correlation between genetic ancestry and socioeconomic and environmental factors that could underlie these differences. More importantly, researchers must avoid the stigmatization of individuals based on perceived or real genetic risks. The latter point will become increasingly sensitive as several 'for profit companies' are offering ancestry and genetic testing directly to consumers and the consequences of the spread of the services of these companies are still unforeseeable.

Introduction to the post-Human Genome Project era, a target for interactions between polygenic and/or multiphenotypical components in cancer control in South America.

Epidemiological studies have suggested that the propensity to develop malignancy involves a complex mix of genetic and environmental determinants, however both older and innovative techniques display unresolved fundamental questions regarding etiology. Current barriers to achieving the potential benefit from this understanding are: 1) incomplete background on the various environmental and genetic factors involved in the carcinogenesis mechanism; 2) difficulties in accurately differentiating specific molecular subtypes and measuring the effective cellular exposure dose; and 3) difficulties in determining the multifactorial interaction between genetic and environmental factors. To extrapolate Human Genome Project research findings to the Post-Human Genome Project era, South America provides a large population and large-pedigree families, thus including genetically heterogeneous and less heterogeneous groups. An initial strategy might be to trace high risk populations and the respective exposures to which they are susceptible, such as: 1) migration, identifying rural migrant populations; 2) inherent susceptibility, studying "long term homogeneous populations" or large families living in similar rural environments; and 3) dissection of gene-environmental interaction