Dcode.org anthology of comparative genomic tools

Comparative genomics provides the means to demarcate functional regions in anonymous DNA sequences. The successful application of this method to identifying novel genes is currently shifting to deciphering the non-coding encryption of gene regulation across genomes. To facilitate the practical application of comparative sequence analysis to genetics and genomics, we have developed several analytical and visualization tools for the analysis of arbitrary sequences and whole genomes. These tools include two alignment tools, zPicture and Mulan; a phylogenetic shadowing tool, eShadow for identifying lineage- and species-specific functional elements; two evolutionary conserved transcription factor analysis tools, rVista and multiTF; a tool for extracting cis-regulatory modules governing the expression of co-regulated genes, Creme 2.0; and a dynamic portal to multiple vertebrate and invertebrate genome alignments, the ECR Browser. Here, we briefly describe each one of these tools and provide specific examples on their practical applications. All the tools are publicly available at the website

A view of evolution by a Christian biologist

This paper, which originated as an address to a gathering of Dutch Reformed Ministers in Potchefstroom, deals with the response of a Christian to concepts of evolution. The paper looks at the concept of “the beginning”, the origin of life , genetics, mutation and natural selection, the origin of diversity or speciation, man as a biological species, the missing link (in which attention is given to various theories), and finally theories of human evolution. These include the neoteny theory, the savannah theory and the aquatic theory. Following a discussion of the various aspects of these theories, the authors go on to a discussion of the evolution of intelligence and culture, and reach the conclusion that “for a Christian, evolution may help him to understand more about God and his love and his work, and also then to have more security in the belief in God”

Effect of age and cytoskeletal elements on the indentation-dependent mechanical properties of chondrocytes.

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15-2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level

Genome-wide de Novo Prediction of Proximal and Distal Tissue-Specific Enhancers

Determining how transcriptional regulatory networks are encoded in the human genome is essential for understanding how cellular processes are directed. Here, we present a novel approach for systematically predicting tissue specific regulatory elements (REs) that blends genome-wide expression profiling, vertebrate genome comparisons, and pattern analysis of transcription factor binding sites. This analysis yields 4,670 candidate REs in the human genome with distinct tissue specificities, the majority of which reside far away from transcription start sites. We identify key transcription factors (TFs) for 34 distinct tissues and demonstrate that tissue-specific gene expression relies on multiple regulatory pathways employing similar, but different cohorts of interacting TFs. The methods and results we describe provide a global view of tissue specific gene regulation in humans, and propose a strategy for deciphering the transcriptional regulatory code in eukaryotes

Optimization of a genomic editing system using CRISPR/Cas9-induced site-specific gene integration

The CRISPR-Cas system is an adaptive immune system found in bacteria which helps protect against the invasion of other microorganisms. This system induces double stranded breaks at precise genomic loci (1) in which repairs are initiated and insertions of a target are completed in the process. This mechanism can be used in eukaryotic cells in combination with sgRNAs (1) as a tool for genome editing. By using this CRISPR-Cas system, in addition to the “safe harbor locus,” ROSAβ26, the incorporation of a target gene into a site that is not susceptible to gene silencing effects can be achieved through few simple steps. PCR amplification of the target genes , ROSA26 and mKate2, with a sgRNA scaffold and T7 promoter followed by in vitro transcription aim to produce an RNA product. This sgRNA product can be run through a digestion with Cas9 to validate cleavage of the genomic ROSA DNA template or mKate plasmid. Osteoblast mouse cells are transfected and labeled through partial uptake by the CRISPR mechanism, by cutting in the ROSA loci and repairing with pieces of the fluorescent mKate2 plasmid. These cells were measured via flow cytometry to give a percentage of red cells. This data shows the scaffolding construct created is targeted by the Cas9 endonuclease and through homologous repair the cells will incorporate the mKate2 target gene in vitro in MC3T3 mouse cells

Nanocomposite Scaffold for Chondrocyte Growth and Cartilage Tissue Engineering: Effects of Carbon Nanotube Surface Functionalization

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold\u27s mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels + SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair

Medicolegal perspectives of interpersonal violence: A review of first-contact clinical notes

Background. South Africa has one of the highest rates of interpersonal violence (IPV), in all its forms, in the world. Incidents of assault are largely under-reported and place an immense burden on the healthcare, fiscal and judicial systems. The first contact a victim of IPV has with a medical practitioner may be the only opportunity to identify, record and preserve valuable evidence, as evidence not captured on this occasion may be lost forever. The accuracy and quality of clinical notes taken at the time may be of paramount importance in facilitating the administration of justice.Objectives. (i) To investigate the adequacy of medicolegal note keeping by doctors in cases where subsequent legal proceedings may ensue; and (ii) to apprise clinicians of their shared responsibility in contributing to the administration of justice through both appropriate patient management and objective and contemporaneous recording of findings from a medicolegal perspective.Methods. A prospective descriptive study was conducted over a period of 18 months from 2016 to 2018. The investigators reviewed patient files and critically appraised first-contact clinical notes in fatal-outcome cases of IPV admitted to the Pretoria Medico-Legal Laboratory during the study period. The cases were reviewed using rubrics specifically designed by the investigators to critically but consistently assess the adequacy of documentation of the medicolegal aspects applicable to each case.Results. One hundred cases met the defined criteria for inclusion in the study. The victims were predominantly male (98%), and most (79%) were aged <40 years. Blunt-force injuries were the most frequent type of injury (43%), while gunshot wounds accounted for 36% of cases and sharp-force injuries were documented in 11%. Insufficient medicolegal documentation, wound description and evidence collection, by medical practitioners, was identified across all wounding modalities in the study sample.Conclusions. This study showed that medicolegal documentation in cases of IPV is suboptimal, with many important parameters not being routinely recorded, which is likely to impact negatively on criminal investigations and downstream legal proceedings. Greater emphasis on these issues is required during the undergraduate training of healthcare workers in a society as severely afflicted by IPV as SA. Although this study focused on fatal-outcome cases, these conclusions are equally applicable to many more cases where investigators, prosecutors and presiding judicial officers may be dependent on findings contemporaneously and objectively recorded by medical professionals.

The role of Data Transfer Agreements in ethically managing data sharing for research in South Africa

A multitude of legislation impacts the use of samples and data for research in South Africa (SA). With the coming into effect of the Protection of Personal Information Act No. 4 of 2013 (POPIA) in July 2021, recent attention has been given to safeguarding research participants' personal information. The protection of participants' privacy in research is essential, but it is not the only risk at stake in the use and sharing of personal information. Other rights and interests that must also be considered and safeguarded include the right to non-discrimination, the right to dignity, and the right to enjoy the benefits of scientific progress. This can be achieved by ensuring that the ethical values and universal principles that underpin the research landscape, namely equity, reciprocity, justice and solidarity, are embedded in the regulatory framework for the management and use of personal information. This paper advocates an integrated bioethics approach to the use of personal information for research in SA. A key feature in embedding such an approach is a Data Transfer Agreement (DTA) that appropriately integrates the safeguards and protections set out by our regulatory framework with bioethical rules and procedures when personal information is used and shared for research purposes. This paper thus calls for the development of a national DTA that is guided by key ethical principles when data are shared for research purposes. This paper will be followed by a webinar on 23 June 2022 hosted by the Department of Science and Innovation and the SA Medical Research Council, where the recommendations and suggestions set out herein will be unpacked, discussed and debated by relevant stakeholders

Unspoken victims: A national study of male rape incidents and police investigations in South Africa

Background. The burden of sexual violence has been well described in children of both sexes and in women, but there is minimal literature on adult male rape victims. Studies of adult male rape victims have mainly been conducted among incarcerated males or military personnel, and in high-income countries.Objectives. To describe the epidemiology, occurrence and reporting of rape cases involving male victims, both child (<18 years old) and adult, in South Africa (SA).Methods. The study consisted of a nationally representative sample of case dockets maintained by the SA Police Service of rape incidents reported in 2012. A retrospective review of the dockets provided sociodemographic information on the victim and suspect, the circumstances of the rape and the medicolegal services provided to the victim. Data on male victims were analysed using Stata 13 to test for significant differences between child and adult male victims.Results. The study comprised 209 male victims, including 120 (57.4%) children and 89 (42.6%) adults. The findings showed that there were significant differences in the occurrence and reporting of rape of male victims by age. Adult males experienced more violent rapes, perpetrators were more likely to be armed and often humiliated the victim, and rapes were more likely to occur in institutional settings. Adult males reported incidents of rape earlier and therefore had visible non-genital injuries during the medical examination. In contrast, more child rapes involved known perpetrators, occurred in a home and perpetrators were more likely to act kindly to the victim after the incident. This parallels the patterns in rape circumstances seen in female adult and child victims.Conclusions. While there is political commitment to understanding sexual violence against women as a societal problem, work on such violence against men lags behind and is little understood. Rape of males needs to be acknowledged, and their vulnerabilities to sexual abuse and rape need to be addressed. Prevention efforts to end violence against women and girls, especially in relation to children, can be used to address violence against men and boys