Technology In Biomedical Research Research Articles

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.

3D Cell Technology in Biomedical Research

Traditional two dimensional cell culture has enabled great strides in biomedicine but needs to be improved to be able to keep up with the demands of modern biomedical research. 2D monolayer culture cannot replicate tissue responses and needs to be supplemented with extensive animal research. Growing cells in three dimensional scaffolds provides a more functional model for biomedical research than traditional monolayer culture. Depending on the needs and the complexity of the model there are several ways that 3D models can be initiated. Simple spheroids can be grown in low adherence plates and in hanging drops while larger spheroids and co-cultured ones need to be grown in systems with greater support such as hydro gels. The system that offers the greatest flexibility is the magnetic levitation approach. In the paper we offer a brief resume to various 3D methods and their characteristics to ease the choice of methods for implementing 3D cell culture techniques.

Tele-Robotic Platform for Dexterous Optical Single-Cell Manipulation.

Single-cell manipulation is considered a key technology in biomedical research. However, the lack of intuitive and effective systems makes this technology less accessible. We propose a new tele–robotic solution for dexterous cell manipulation through optical tweezers. A slave-device consists of a combination of robot-assisted stages and a high-speed multi-trap technique. It allows for the manipulation of more than 15 optical traps in a large workspace with nanometric resolution. A master-device (6+1 degree of freedom (DoF)) is employed to control the 3D position of optical traps in different arrangements for specific purposes. Precision and efficiency studies are carried out with trajectory control tasks. Three state-of-the-art experiments were performed to verify the efficiency of the proposed platform. First, the reliable 3D rotation of a cell is demonstrated. Secondly, a six-DoF teleoperated optical-robot is used to transport a cluster of cells. Finally, a single-cell is dexterously manipulated through an optical-robot with a fork end-effector. Results illustrate the capability to perform complex tasks in efficient and intuitive ways, opening possibilities for new biomedical applications.

World Health Organization Report: Current Crisis of Antibiotic Resistance

Antibiotic resistance is the most challenging clinical and public health problem. Despite of living in the era of novel technologies in biomedical research, many of untreatable infectious diseases are ranked as the main causes of human death worldwide. Increased antibiotic use in human and use in animal production are the two major causes of emergence of resistant bacteria in hospitals, human communities, and also animal farms. Current body of evidences is indicating that major factors that led to existing crisis on antibiotics worldwide are poor educational programs on hygiene and health, inappropriate prescription in addition to the overprescription in clinical settings (mainly in developing countries with easier access to the antibiotics) and lack of accurate diagnostic tools in laboratories in order to control the emergence of antibiotics against widely used drugs in community. It sounds using the antibodies against problematic bacteria in farms has more benefits than treating them with susceptible antibiotics. As best strategy, we pointed that the crisis of antibiotic resistance may be solved when all contributors be acknowledged to their responsibilities and duties to minimize this global problem threatening the human health. China and the USA as the two main antibiotics user in industrial scale should have taken new policy in meat industry. Currently, antibiotic resistance presents a growing health threat worldwide being the cause of many nosocomial and often deadly infections.

BSSE: An open-source image processing tool for miniaturized microscopy

Single-photon-excitation-based miniaturized microscope, or miniscope, has recently emerged as a powerful tool for imaging neural ensemble activities in freely moving animals. In the meanwhile, this highly flexible and implantable technology promises great potential for studying a broad range of cells, tissues and organs. To date, however, applications have been largely limited by the properties of the imaging modality. It is therefore highly desirable for a method generally applicable for processing miniscopy images, enabling and extending the applications to diverse anatomical and functional traits, spanning various cell types in the brain and other organs. We report an image processing approach, termed BSSE, for background suppression and signal enhancement for miniscope image processing. The BSSE method provides a simple, automatic solution to the intrinsic challenges of overlapping signals, high background and artifacts in miniscopy images. We validated the method by imaging synthetic structures and various biological samples of brain, tumor, and kidney tissues. The work represents a generally applicable tool for miniscopy technology, suggesting broader applications of the miniaturized, implantable and flexible technology for biomedical research.

Quantifying CRISPR off-target effects.

Recent advances in the era of genetic engineering have significantly improved our ability to make precise changes in the genomes of human cells. Throughout the years, clinical trials based on gene therapies have led to the cure of diseases such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID) and Wiskott-Aldrich syndrome. Despite the success gene therapy has had, there is still the risk of genotoxicity due to the potential oncogenesis introduced by utilising viral vectors. Research has focused on alternative strategies like genome editing without viral vectors as a means to reduce genotoxicity introduced by the viral vectors. Although there is an extensive use of RNA-guided genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein-9 (Cas9) technology for biomedical research, its genome-wide target specificity and its genotoxic side effects remain controversial. There have been reports of on- and off-target effects created by CRISPR-Cas9 that can include small and large indels and inversions, highlighting the potential risk of insertional mutagenesis. In the last few years, a plethora of in silico, in vitro and in vivo genome-wide assays have been introduced with the sole purpose of profiling these effects. Here, we are going to discuss the genotoxic obstacles in gene therapies and give an up-to-date overview of methodologies for quantifying CRISPR-Cas9 effects.

Exploring new technologies in biomedical research

The Health Law, Policy & Ethics Project at Emory University School of Law and the Human Toxicology Project Consortium of the Humane Society of the United States co-sponsored a symposium on October 23, 2017, to showcase innovations using human-based in silico and in vitro models for drug and device discovery. The goal of the symposium was to introduce researchers and students to exciting new tools and possible future careers that will increase understanding of disease and improve the search for effective therapeutics, while reducing reliance on animal testing. The symposium concluded with a discussion between scientists and lawyers about the legal regulation of new biomedical research technologies.

Polymerase-Extension-Based Selection Method for DNA-Encoded Chemical Libraries against Nonimmobilized Protein Targets.

DNA-encoded chemical libraries (DELs) have become an important ligand discovery technology in biomedical research and drug discovery. DELs can be comprised of hundreds of millions to billions of candidate molecules and provide outstanding chemical diversity for discovering novel ligands and inhibitors for a large variety of biological targets. However, in most cases, DELs are selected against purified and immobilized proteins based on binding affinity. The development and application of DELs to more complex biological targets requires selection methods compatible with nonimmobilized and unpurified proteins. Here, we describe an approach using polymerase-based extension and target-directed photo-cross-linking and its application to the interrogation of a solution-phase protein target, carbonic anhydrase II.

Research progress on non-viral vectors of CRISPR/Cas9 system

In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system has become one of the most powerful technologies in biomedical research and shown great potentials in the establishment of disease models as well as the development of new treatments. However, efficient delivery of CRISPR/Cas9 systems to target organs and cells remains challenging. With the rapid development of non-viral vectors, nanocarriers based on liposomes, polymers, and inorganic nanoparticles have shown great potentials for the delivery of CRISPR/Cas9 systems. In this review, we first summarize the potential of the CRISPR/Cas9 system in disease modeling and disease treatment, as well as the limitations. The advantages and disadvantages of delivering the CRISPR/Cas9 system in the form of plasmid DNA, mRNA or protein are then analyzed and the non-viral vectors that have emerged are reviewed. Finally, we summarize the key barriers currently faced by non-viral vectors in the delivery of CRISPR/Cas9 systems and propose strategies that are expected to overcome them.

Bioluminescent imaging: new opportunities

Modern biomedical research technologies actively use bioimaging for studying cells, tissues and whole organisms. Multicolor bioimaging is applied when simultaneous observation of different events at the molecular and cellular level is needed. Bioluminescent imaging methods are the most sensitive, however, their use for multicolor labeling is complicated due to the insufficient number of available uciferin-luciferase pairs. Having a number of advantages compared to previously studied bioluminescent systems, the new bioluminescence systems of higher fungi and marine polychaete Odontosyllis could become a useful expansion of the bioimaging toolbox.

Companion Animals as Technologies in Biomedical Research

In this paper we examine the use of companion animals (pets) in studies of drugs and devices aimed at human and animal health and situate it within the context of philosophy of technology. We argue...

Simultaneous positron emission tomography and ultrafast ultrasound for hybrid molecular, anatomical and functional imaging.

Positron emission tomography-computed tomography (PET-CT) is the most sensitive molecular imaging modality, but it does not easily allow for rapid temporal acquisition. Ultrafast ultrasound imaging (UUI)-a recently introduced technology based on ultrasonic holography-leverages frame rates of up to several thousand images per second to quantitatively map, at high resolution, haemodynamic, biomechanical, electrophysiological and structural parameters. Here, we describe a pre-clinical scanner that registers PET-CT and UUI volumes acquired simultaneously and offers multiple combinations for imaging. We demonstrate that PET-CT-UUI allows for simultaneous images of the vasculature and metabolism during tumour growth in mice and rats, as well as for synchronized multi-modal cardiac cine-loops. Combined anatomical, functional and molecular imaging with PET-CT-UUI represents a high-performance and clinically translatable technology for biomedical research.

Germline and somatic variant identification using BGISEQ-500 and HiSeq X Ten whole genome sequencing.

Technological innovation and increased affordability have contributed to the widespread adoption of genome sequencing technologies in biomedical research. In particular large cancer research consortia have embraced next generation sequencing, and have used the technology to define the somatic mutation landscape of multiple cancer types. These studies have primarily utilised the Illumina HiSeq platforms. In this study we performed whole genome sequencing of three malignant pleural mesothelioma and matched normal samples using a new platform, the BGISEQ-500, and compared the results obtained with Illumina HiSeq X Ten. Germline and somatic, single nucleotide variants and small insertions or deletions were independently identified from data aligned human genome reference. The BGISEQ-500 and HiSeq X Ten platforms showed high concordance for germline calls with genotypes from SNP arrays (>99%). The germline and somatic single nucleotide variants identified in both sequencing platforms were highly concordant (86% and 72% respectively). These results indicate the potential applicability of the BGISEQ-500 platform for the identification of somatic and germline single nucleotide variants by whole genome sequencing. The BGISEQ-500 datasets described here represent the first publicly-available cancer genome sequencing performed using this platform.

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research.

Gene editing in mouse zygotes using the CRISPR/Cas9 system.

The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.

The role of scientific self‐regulation for the control of genome editing in the human germline

CRISPR‐Cas9‐based gene‐editing technology has fuelled a debate about the implications of the possibility of genome editing in the human germline [1], [2]. Scientists have suggested how this technology could be regulated and evaluated to prevent misuse or abuse. Such discussions amongst scientists are not new: in the early days of recombinant DNA technology, scientists called attention to the potential benefits and risks of this new tool to genetically modifying living organisms. This development led to the famous conference held at Asilomar State Beach in California, in 1975. Forty years later, scientists and experts from ethics and law convened in Napa Valley, California, to discuss the scientific, societal and ethical implications of applying genome editing technologies in the human germline. The Napa group explicitly regarded their meeting as being in the tradition of Asilomar, to discuss guidelines and self‐regulation for biotechnological developments. > Emerging technologies in biomedical research often touch on ethical and societal questions that cannot be answered by scientists alone Emerging technologies in biomedical research often touch on ethical and societal questions that cannot be answered by scientists alone. Thus, the inclusion of other experts in discussions about regulation and public engagement [3] has become increasingly important to take into account the wide spectrum of implications; and for maintaining public confidence in the regulatory instruments to control the use of novel technologies. Here, we discuss the opportunities and limitations of self‐regulation for emerging biotechnologies based on a comparison of the Asilomar conference and the Napa meeting and propose a model that combines self‐regulation with national and international governance in the context of using gene‐editing technologies in the human germline. ### From Asilomar to Napa The paradigm case of scientific self‐regulation in the biosciences is the above‐mentioned “Asilomar Conference on Recombinant DNA”, which was held after restriction enzymes and …

Encapsulation of Insulin-Secreting Cells Expressing a Genetically Encoded Fluorescent Calcium Indicator for Cell-Based Sensing In Vivo.

The development of cell-based biosensors that give insight into cell and tissue function in vivo is an attractive technology for biomedical research. Here, the development of a cell line expressing a fluorescent calcium sensor for the study of beta-cell function in vivo is reported. The bioresponsive cell model is based on INS-1E pancreatic beta-cells, stably expressing the genetically encoded cameleon-based fluorescent sensor YC3.6cyto . Following single-cell selection and expansion, functional testing and in vitro encapsulation experiments are used to identify a suitable clone of INS-1E cells expressing the calcium sensor. This clone is transplanted subcutaneous in mouse using a cell macroencapsulation system based on flat sheet porous membranes. Cells in the implanted capsules are able to respond to glucose in vivo by secreting insulin and thereby contributing to the regulation of glycaemia in the mice. Furthermore, fluorescence imaging of explanted devices shows that encapsulated cells maintain high level expression of YC3.6cyto in vivo. In conclusion, these data show that encapsulated INS-1E cells stably expressing a genetically encoded calcium sensor can be successfully implanted in vivo, and therefore serve as biosensing element or in vivo model to longitudinally monitor the function of pancreatic beta-cells.

Genome Editing in Mice Using TALE Nucleases.

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

PROPER: Performance visualization for optimizing and comparing ranking classifiers in MATLAB.

BackgroundOne of the recent challenges of computational biology is development of new algorithms, tools and software to facilitate predictive modeling of big data generated by high-throughput technologies in biomedical research.ResultsTo meet these demands we developed PROPER - a package for visual evaluation of ranking classifiers for biological big data mining studies in the MATLAB environment.ConclusionPROPER is an efficient tool for optimization and comparison of ranking classifiers, providing over 20 different two- and three-dimensional performance curves.Electronic supplementary materialThe online version of this article (doi:10.1186/s13029-015-0047-1) contains supplementary material, which is available to authorized users.

The ethics of citizen science in biomedicine

Background: Research in biomedicine and healthcare increasingly includes participatory initiatives and harnesses direct contributions from lay citizens in terms of data, samples, expertise and cognitive capacities. Many such practices are called 'citizen science' (CS). In our research project, (1) we explore the diversity of CS initiatives in biomedicine, including their underlying motivations and their main typologies; (2) describe and discuss emerging ethical challenges due to CS. Methodology: On the basis of a literature and websites review, we identified “stakes” associated with CS, i.e. reasons that explain why CS may lead to better biomedical science and elaborate a typology of initiatives. We then mapped each of these categories to an established framework of research ethics, the seven principles described by Emanuel et al. (2000). Results: We found that CS initiatives are seen as facilitating the uptake of network IT technologies in biomedical research and data-intensive research projects (esp. in genomics), and promote the establishment of infrastructure for a more precise and personalized medicine. CS is also seen as heralding the values of openness of peer-to-peer movements, turn innovation and discovery pipelines in “open systems” and reduce costs. Learning opportunities, empowerment of patients/citizens and citizen engagement in science also appear prominently among motivations for CS. There are few cases of genuinely participant-led research projects, however a wider variety of actors participates to such initiatives, including data scientists and biotech entrepreneurs. We found that CS initiatives rise novel ethical concerns regarding the assessment of societal value of research, informed consent, data privacy, and risks for participants, along with governance challenges, especially in genuinely participant-led projects. Discussion: By describing our research pipeline and our preliminary results, we propose that social science analysis and ethical reflection should be combined to study emerging trends in health systems and to propose actionable solutions for healthcare and biomedicine.