Because there is no widely used software for analyzing RNA-seq data that has a graphical user interface, this protocol provides an example of analyzing microarray data using Babelomics. This analysis entails performing quantile normalization and then detecting differentially expressed genes associated with the transgenesis of a human oncogene c-Myc in mice. Finally, hierarchical clustering is performed on the differentially expressed genes using the Cluster program, and the results are visualized using TreeView.
This protocol describes mapping short sequence reads to a reference genome using several programs. The example in this protocol starts with a ChIP-seq data set in FASTQ format, aligns the reads to the human genome using Bowtie, and uses some useful utilities of SAMtools and BEDTools. SAMtools and BEDTools are two collections of executables for manipulating the results of short-read aligners. By combining these tools, one can summarize and visualize alignments produced by Bowtie and perform basic analysis, such as determining the number of reads that are mapped to a certain gene. These tools can also be easily incorporated into computational pipelines of more complex analyses.
Designing oligonucleotide primers is a crucial step for successful molecular biology experiments that require the use of the polymerase chain reaction (PCR). PCR involves cycles of three steps: denaturation, annealing, and extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are separated into single strands. During annealing, a pair of primers is annealed to the complementary regions of the single-stranded molecules. In the extension step, DNA polymerase extends the primers to produce DNA molecules that correspond to the region bracketed by the primers (the amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is 94 degrees C, 60 degrees C, and 70 degrees C, respectively. Poorly designed primers may lead to no amplification product or additional undesired amplified fragments. The goals of primer design include good primer specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the prevention of primer hairpins or primer dimers.
We report the performance of our protein-protein docking pipeline, including the ZDOCK rigid-body docking algorithm, on 19 targets in CAPRI rounds 28-34. Following the docking step, we reranked the ZDOCK predictions using the IRAD scoring function, pruned redundant predictions, performed energy landscape analysis, and utilized our interface prediction approach RCF. In addition, we applied constraints to the search space based on biological information that we culled from the literature, which increased the chance of making a correct prediction. For all but two targets we were able to find and apply biological information and we found the information to be highly accurate, indicating that effective incorporation of biological information is an important component for protein-protein docking. Proteins 2016.
With the rapid accumulation of publicly available small RNA sequencing datasets, third-party meta-analysis across many datasets is becoming increasingly powerful. Although removing the 3 adapter is an essential step for small RNA sequencing analysis, the adapter sequence information is not always available in the metadata. The information can be also erroneous even when it is available. In this study, we developed DNApi, a lightweight Python software package that predicts the 3 adapter sequence de novo and provides the user with cleansed small RNA sequences ready for down stream analysis. Tested on 539 publicly available small RNA libraries accompanied with 3 adapter sequences in their metadata, DNApi shows near-perfect accuracy (98.5%) with fast runtime (~2.85 seconds per library) and efficient memory usage (~43 MB on average). In addition to 3 adapter prediction, it is also important to classify whether the input small RNA libraries were already processed, i.e. the 3 adapters were removed. DNApi perfectly judged that given another batch of datasets, 192 publicly available processed libraries were "ready-to-map" small RNA sequence. DNApi is compatible with Python 2 and 3, and is available at https://github.com/jnktsj/DNApi. The 731 small RNA libraries used for DNApi evaluation were from human tissues and were carefully and manually collected. This study also provides readers with the curated datasets that can be integrated into their studies.
This protocol illustrates the steps of searching a sequence database with BLAST, which in essence performs pairwise alignment between the query sequence and each target sequence in the database. Steps for performing multiple sequence alignments with ClustalW are then described. Argonaute 2 (Ago2) is an essential component for small interfering RNA (siRNA)-directed RNA interference (RNAi) in fruit flies. This protocol uses Ago2 to demonstrate how to search for all Ago2 paralogs in the fly proteome using BLAST and to obtain a multiple sequence alignment of these sequences with ClustalW.
Genomic data and annotations are rapidly accumulating in databases such as the UCSC Genome Browser, NCBI, and Ensembl. Given the massive scale of these genomic databases, it is important to be able to easily retrieve known data and annotations of a specified genomic locus. For example, for a newly identified cis-regulatory element bound by a transcription factor, questions that immediately come to mind include whether the element is near a transcriptional start site and, if so, the name of the corresponding gene, and whether the histones or DNA at the locus are modified. The UCSC Genome Browser organizes data and annotations (called tracks) around the reference sequences or draft assemblies of many eukaryotic genomes and presents them using a powerful web-based graphical interface. This protocol describes how to use the UCSC Genome Browser to visualize selected tracks at specified genomic regions, download the data and annotations for further analysis, and retrieve multiple sequence alignments and their conservation scores.
Bioinformatics was brought into the spotlight in the late 1990s through the Human Genome Project. With the rapid accumulation of completed genomes, it was soon realized that for the vast majority of the newly identified genes and other functional regions of the genomes there were no other biological data. One way of inferring biological function is through homology: Because homologous genes have a common evolutionary descent, they are likely to have the same biological function. A large number of bioinformatics tools have been designed for rapidly and accurately comparing sequences of genes or proteins, comparing gene sequences with genomes, and comparing genomes. Two widely used tools for sequence alignment and homology searches, BLAST and ClustalW, are introduced here.
A generalized framework for computational design and mutational scanning of T-cell receptor binding interfaces
T-cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for cancer where they are the key components of new cellular therapies as well as soluble biologics. Many studies have generated high affinity TCRs in order to enhance sensitivity. Recent outcomes, however, have suggested that fine manipulation of TCR binding, with an emphasis on specificity may be more valuable than large affinity increments. Structure-guided design is ideally suited for this role, and here we studied the generality of structure-guided design as applied to TCRs. We found that a previous approach, which successfully optimized the binding of a therapeutic TCR, had poor accuracy when applied to a broader set of TCR interfaces. We thus sought to develop a more general purpose TCR design framework. After assembling a large dataset of experimental data spanning multiple interfaces, we trained a new scoring function that accounted for unique features of each interface. Together with other improvements, such as explicit inclusion of molecular flexibility, this permitted the design new affinity-enhancing mutations in multiple TCRs, including those not used in training. Our approach also captured the impacts of mutations and substitutions in the peptide/MHC ligand, and recapitulated recent findings regarding TCR specificity, indicating utility in more general mutational scanning of TCR-pMHC interfaces.
BACKGROUND and AIMS: It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing.
METHODS: We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be upregulated following tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines.
RESULTS: We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS upregulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 mRNA or high levels of HMGA2 mRNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of MAPK phosphorylation.
CONCLUSIONS: Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via MAPK, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.
Rapid development and commercialization of instruments that can accurately, rapidly, and cheaply sequence billions of DNA bases is revolutionizing molecular biology and medicine. Because a reference genome is usually available, the first bioinformatics challenge presented by the new generation of high-throughput sequencers is the genome mapping problem, where each read is mapped to a reference genome to reveal its location(s). An introduction to mapping algorithms, as well as factors that influence their results, is provided here.
The three-dimensional organization of the human genome is non-random in interphase cells. Heterochromatin is highly clustered at the nuclear periphery, adjacent to nucleoli, and near centromeres. These localizations are reshuffled during mitosis when the chromosomes are condensed, nucleoli disassembled, and the nuclear envelope broken down. After cytokinesis, heterochromatin is re-localized to the domains described above. However, the mechanisms by which this localization is coordinated are not well understood. This dissertation will present evidence showing that both CAF-1 p150 and Ki-67 regulate nuclear structure throughout the human cell cycle.
Chromatin Assembly Factor 1 (CAF-1) is a highly conserved three-subunit protein complex which deposits histones (H3/H4)2 heterotetramers onto replicating DNA during S-phase of the cell cycle. The N-terminal domain of the largest subunit of CAF-1 (p150N) is dispensable for histone deposition, and instead regulates the localization of specific loci (Nucleolar-Associated Domains, or “NADs”) and several proteins to the nucleolus during interphase. One of the proteins regulated by p150N is Ki-67, a protein widely used as a clinical marker of cellular proliferation. Depletion of Ki-67 decreases the association of NADs to the nucleolus in a manner similar to that of p150. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins that surrounds all condensed chromosomes during mitosis. A subset of p150 localizes to the PCL during mitosis, and depletion of p150 disrupts Ki-67 localization to the PCL. This activity was mapped to the Sumoylation Interacting Motif (SIM) within p150N, which is also required for the localization of NADs and Ki-67 to the nucleolus during interphase. Together, these studies indicate that p150N coordinates the three-dimensional arrangement of both interphase and mitotic chromosomes via Ki-67.
Histone Deacetylase 3 Coordinates Heart Development Through Stage-Specific Roles in Cardiac Progenitor Cells
Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.
A Translational Pathway for Recombinant Adeno-Associated Virus Human Gene Therapy: From Target Identification and Animal Modeling of the Disease to Non-Human Primate and Human Studies
Many steps go into developing a clinical viral gene therapy. The course starts with appropriate disease selection and moves through the many hurdles of in-vitro testing, animal model validation and proof-of-concept studies, all the way through pre-clinical large animal studies. In this thesis, I propose to outline the process of developing a translation pathway for a gene therapy using recombinant adeno-associated virus (rAAV). I will expand on this outline using data that I have generated during the course of my Ph.D. that ranges from animal model validation all the way through pre-clinical vector stability studies. Two disease models will be discussed throughout this thesis, Cockayne Syndrome (CS) and Alpha-1 Antitrypsin Deficiency (AATD). Cockayne Syndrome is a rare autosomal recessive genetic disorder involving mutations in either the CSA or CSB gene, leading to defects in DNA repair. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, which leads to mental retardation, post-natal growth defects, ocular abnormalities, and shortened life expectancy. Alpha-1 antitrypsin is a serine protease inhibitor largely produced in the liver that mainly functions to inhibit neutrophil elastase within the lung. AATD leads to an increased risk of emphysema, with shortened life expectancy, and also results in accumulations of mutant AAT polymers in the liver, sometimes leading to liver failure. Using these two disease models I will outline the upstream and downstream pre-clinical work as well as the transition to clinical trials of a rAAV based gene therapy.
This dataset is the primary data source for a manuscript submitted for publication.
In many states, outdated rules and regulations restrict nurse practitioners (NPs) from practicing to their full potential, often limiting patients’ access to primary care. Modernizing NP state scope of practice laws and allowing patients greater access to NPs services is a priority. Unlike other professions, nurse practitioners have been unable to consistently influence legislative changes to health policy. This study examined the political efficacy and participation of nurse practitioners in the United States today (N=632). A descriptive cross sectional design, in conjunction with a political efficacy framework, evaluated nurse practitioners’ participation in political activities and their internal and external political efficacy. Increased internal political efficacy was significantly (p < 0.001) associated with NPs who were older, had specific health policy education, and have been mentored in health policy. Our findings show that NPs vote at consistently higher rates (94%) than the general population and almost 50% report contacting legislators via mail/email/phone. As a group however, NPs report limited participation in other political activities, especially grassroots efforts. These findings hold significant implications for the profession as we strive to make policy changes across the country. It is important that educators assess our current methods of educating NPs about politics and health policy. Professional organizations and policy makers must reexamine outreach and strategies to inspire greater grassroots engagement of NPs.
Macrocognition in the Health Care Built Environment (m-HCBE): A Focused Ethnographic Study of 'Neighborhoods' in a Pediatric Intensive Care Unit: A Dissertation
Objectives: The objectives of this research were to describe the interactions (formal and informal) in which macrocognitive functions occur and their location on a pediatric intensive care unit (PICU); describe challenges and facilitators of macrocognition using three constructs of space syntax (openness, connectivity, and visibility); and analyze the health care built environment (HCBE) using those constructs to explicate influences on macrocognition.
Background: In high reliability, complex industries, macrocognition is an approach to develop new knowledge among interprofessional team members. Although macrocognitive functions have been analyzed in multiple health care settings, the effect of the HCBE on those functions has not been directly studied. The theoretical framework, “Macrocognition in the Health Care Built Environment” (m-HCBE) addresses this relationship.
Methods: A focused ethnographic study was conducted, including observation and focus groups. Architectural drawing files used to create distance matrices and isovist field view analyses were compared to panoramic photographs and ethnographic data.
Results: Neighborhoods comprised of corner configurations with maximized visibility enhanced team interactions as well as observation of patients, offering the greatest opportunity for informal situated macrocognitive interactions (SMIs).
Conclusions: Results from this study support the intricate link between macrocognitive interactions and space syntax constructs within the HCBE. These findings help to advance the m-HCBE theory for improving physical space by designing new spaces or refining existing spaces, or for adapting IPT practices to maximize formal and informal SMI opportunities; this lays the groundwork for future research to improve safety and quality for patient and family care.
Undocumented college students face several barriers that may place them at high risk of poor mental health. Despite growing up and receiving primary and secondary (K-12) education in the U.S., many undocumented young adults cannot legally work, vote or drive in most states. Their illegal status interferes with their ability to accumulate relevant/ practical work experience leading to the inability to develop the necessary job skills before graduating high school, which can limit their employment opportunities.
Case Diagnosis: Our patient experienced worsening left foot neuropathy following chemotherapy and radiation treatment for sarcoma.
Case Description: A 24-year-old man underwent local resection of a 12cm x 8cm x 14.5cm rhabdomyosarcoma in the left vastus lateralis. Then, he was treated with vincristine for 40 weeks and radiation to the left lateral thigh with a maximum dose of 50.4 Gy. The sciatic nerve was outside the target area and received a lower dose. While undergoing chemotherapy, the patient experienced bilateral dysesthesias in his fingertips and feet. He had no history of neuropathy prior to treatment. After chemotherapy was completed, these symptoms subsided in all extremities except the left foot, which developed atraumatic plantar flexion and dorsiflexion weakness, great toe extensor and flexor weakness, decreased sensation in the distal left toe to the metatarsal. Electromyography and needle conduction studies demonstrated left worse than right polyneuropathy mainly affecting the tibial and peroneal motor nerves. There was no clear evidence of a single nerve compressive lesion and repeat scans of the thigh showed no new lesion. Given the presence of milder nerve abnormalities on the right in addition to left sided weakness, the cause is likely multifactorial and temporally related to cancer treatments.
Discussions: Persistent or worsening features may appear in patients who received vincristine despite termination of treatment. The pattern is typically sensorimotor; however, this patient demonstrates mainly motor abnormalities. The left worse than right pattern could suggest radiation-induced neuropathy, but no myokymic potentials were seen. Myokymic potentials are common in radiation neuropathy, although their absence does not rule it out. Treatment included physical therapy, gabapentin, and an ankle foot orthosis.
Conclusions: Fourteen months after completing radiation and seven months after completing chemotherapy (seven months after symptom onset), the patient’s symptoms are markedly improved. This case demonstrates that neuropathy after treatment in sarcoma patients may be multifactorial.
Objectives: Pre-operative physical therapy has been shown to reduce post-acute care service utilization. Shifting rehabilitation to the presurgical period, referred to as prehabilitation, could result in reduced recovery time and cost. Limited access to physical therapy may prevent patients from achieving the benefits, and a standard set of independent exercises may be an alternative. We aim to assess the feasibility of an independent exercise program as a pre-surgical intervention for total hip and knee replacement.
Design: Participants were taught two exercises for hip or knee arthritis at least one week prior to surgery and instructed to perform them independently at home. Subjects were contacted three days to one month post-operatively and surveyed about discharge, frequency of exercise, and living status of alone or with others. No adverse effects were reported. Additional information was collected from the subjects’chart including age, BMI, and sex. Discharge outcomes were compared with pre-existing independent factors using univariate and multivariate analyses.
Results: A total of 80 subjects were followed with a home discharge rate of 78.75%. Univariate analyses showed that the presence of other people in the home showed a slight, but non-significant, association with differences of discharge destination. 82.1%-83.3% of patients who live with others were discharged home versus 57.1% of patients living alone (LR chi-square: 3.84, p=0.15). Multivariate analyses showed a slight, but non-significant, association between frequency of prehabilitation and discharge destination (OR=1.212; 95% CI, 0.960-1.530). BMI showed no associated difference in discharge destination.
Conclusions: Increased frequency of prehabilitation and presence of others at home showed slight associations with increased discharges to home, but were non-significant. Increased exposure to prehabilitation (duration times frequency) trends toward more frequent home discharge. Independently performed prehabilitation may be offered as an alternative pre-surgical intervention with likely little to no adverse effect. Larger numbers are needed to determine likelihood of discharge home.