Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the ...CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mitochondrial division is critical for maintenance of mitochondrial morphology and cellular homeostasis. Previous studies have suggested that the mitochondria-ER-cortex anchor (MECA), a tripartite ...membrane contact site between mitochondria, the ER, and the plasma membrane, is involved in mitochondrial division. However, its role is poorly understood. We developed a system to control MECA formation and depletion, which allowed us to investigate the relationship between MECA-mediated contact sites and mitochondrial division. Num1 is the protein that mediates mitochondria-ER-plasma membrane tethering at MECA sites. Using both rapamycin-inducible dimerization and auxin-inducible degradation components coupled with Num1, we developed systems to temporally control the formation and depletion of the native contact site. Additionally, we designed a regulatable Num1-independant mitochondria-PM tether. We found that mitochondria-PM tethering alone is not sufficient to rescue mitochondrial division and that a specific feature of Num1-mediated tethering is required. This study demonstrates the utility of systems that regulate contact-site formation and depletion in studying the biological functions of membrane contact sites.
The mitochondria-ER-cortex anchor (MECA) forms a membrane tether between mitochondria, the endoplasmic reticulum, and the plasma membrane. Multiple functions have been attributed to MECA, including a poorly defined role in mitochondrial division. The authors developed systems to control the formation and depletion of the contact site. Using these tools, they found that mitochondrial tethering alone is not sufficient to rescue mitochondrial division and that a specific feature of MECA-mediated tethering is required. Regulatable contact sites, like those developed in this study, provide the ability to observe the downstream consequences of contact site formation and depletion in real time.
The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, ...interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.
Fragrances and malodors are ubiquitous in the environment, arising from natural and artificial processes, by the generation of volatile organic compounds (VOCs). Although VOCs constitute only a ...fraction of the metabolites produced by an organism, the detection of VOCs has a broad range of civilian, industrial, military, medical, and national security applications. The VOC metabolic profile of an organism has been referred to as its 'volatilome' (or 'volatome') and the study of volatilome/volatome is characterized as 'volatilomics', a relatively new category in the 'omics' arena. There is considerable literature on VOCs extracted destructively from microalgae for applications such as food, natural products chemistry, and biofuels. VOC emissions from living (in vivo) microalgae too are being increasingly appreciated as potential real-time indicators of the organism's state of health (SoH) along with their contributions to the environment and ecology. This review summarizes VOC emissions from in vivo microalgae; tools and techniques for the collection, storage, transport, detection, and pattern analysis of VOC emissions; linking certain VOCs to biosynthetic/metabolic pathways; and the role of VOCs in microalgae growth, infochemical activities, predator-prey interactions, and general SoH.
Integration of catalytic nanostructured platinum and palladium within 3D microscale structures or fluidic environments is important for systems ranging from micropumps to microfluidic chemical ...reactors and energy converters. We report a straightforward procedure to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. These materials display excellent catalytic, electrical, and electrochemical properties, and we demonstrate high-resolution integration of catalysts within 3D defined microenvironments to generate directed autonomous particle and fluid transport.
The increasing incidence of cardiovascular disease (CVD) has led to a significant ongoing need to address this surgically through coronary artery bypass grafting (CABG) and percutaneous coronary ...interventions (PCI). From this, there continues to be a substantial burden of mortality and morbidity due to complications arising from endothelial damage, resulting in restenosis. Whilst mast cells (MC) have been shown to have a causative role in atherosclerosis and other vascular diseases, including restenosis due to vein engraftment; here, we demonstrate their rapid response to arterial wire injury, recapitulating the endothelial damage seen in PCI procedures. Using wild‐type mice, we demonstrate accumulation of MC in the femoral artery post‐acute wire injury, with rapid activation and degranulation, resulting in neointimal hyperplasia, which was not observed in MC‐deficient KitW‐sh/W‐sh mice. Furthermore, neutrophils, macrophages, and T cells were abundant in the wild‐type mice area of injury but reduced in the KitW‐sh/W‐sh mice. Following bone‐marrow‐derived MC (BMMC) transplantation into KitW‐sh/W‐sh mice, not only was the neointimal hyperplasia induced, but the neutrophil, macrophage, and T‐cell populations were also present in these transplanted mice. To demonstrate the utility of MC as a target for therapy, we administered the MC stabilizing drug, disodium cromoglycate (DSCG) immediately following arterial injury and were able to show a reduction in neointimal hyperplasia in wild‐type mice. These studies suggest a critical role for MC in inducing the conditions and coordinating the detrimental inflammatory response seen post‐endothelial injury in arteries undergoing revascularization procedures, and by targeting the rapid MC degranulation immediately post‐surgery with DSCG, this restenosis may become a preventable clinical complication.
Acute arterial wire injury in mice triggers an inflammatory cascade mediated through mast cell degranulation resulting in inflammatory cell infiltration and ultimately, neointimal hypertrophy of the artery. Disodium cromoglycate (DSCG) inhibits mast cell degranulation, resulting in decreased inflammatory cell infiltration and reduced neointimal hypertrophy, demonstrating that DSCG may be a good candidate as a therapeutic following arterial surgical intervention.
In order to design hybrid cellular/synthetic devices such as sensors and vaccines, it is important to understand how the metabolic state of living cells changes upon physical confinement within ...three-dimensional (3D) matrices. We analyze the gene expression patterns of stationary phase Saccharomyces cerevisiae (S. cerevisiae) cells encapsulated within three distinct nanostructured silica matrices and relate those patterns to known naturally occurring metabolic states. Silica encapsulation methods employed were lipid-templated mesophase silica thin films formed by cell-directed assembly (CDA), lipid-templated mesophase silica particles formed by spray drying (SD), and glycerol-doped silica gel monoliths prepared from an aqueous silicate (AqS+g) precursor solution. It was found that the cells for all three-encapsulated methods enter quiescent states characteristic of response to stress, albeit to different degrees and with differences in detail. By the measure of enrichment of stress-related gene ontology categories, we find that the AqS+g encapsulation is more amenable to the cells than CDA and SD encapsulation. We hypothesize that this differential response in the AqS+g encapsulation is related to four properties of the encapsulating gel: (1) oxygen permeability, (2) relative softness of the material, (3) development of a protective sheath around individual cells (visible in TEM micrographs vide infra), and (4) the presence of glycerol in the gel, which has been previously noted to serve as a protectant for encapsulated cells and can serve as the sole carbon source for S. cerevisiae under aerobic conditions. This work represents a combination of experiment and analysis aimed at the design and development of 3D encapsulation procedures to induce, and perhaps control, well-defined physiological behaviors.
Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in ...amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial ...adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.
•Cysteine linker modified AcrIIA4 retains the ability to bind and inhibit the CRISPR/Cas9 RNP.•Surface immobilized AcrIIA4 binds Cas9 and Cas9-RNP with high specificity.•Detect Cas9 RNP in whole cell extracts at a limit of detection of 8 nm using electrochemical methods.
Racial disparities in cancer incidence and outcomes are well-documented in the US, with Black people having higher incidence rates and worse outcomes than White people. In this review, we present a ...summary of almost 30 years of research conducted by investigators at the Karmanos Cancer Institute’s (KCI’s) Population Studies and Disparities Research (PSDR) Program focusing on Black-White disparities in cancer incidence, care, and outcomes. The studies in the review focus on individuals diagnosed with cancer from the Detroit Metropolitan area, but also includes individuals included in national databases. Using an organizational framework of three generations of studies on racial disparities, this review describes racial disparities by primary cancer site, disparities associated with the presence or absence of comorbid medical conditions, disparities in treatment, and disparities in physician-patient communication, all of which contribute to poorer outcomes for Black cancer patients. While socio-demographic and clinical differences account for some of the noted disparities, further work is needed to unravel the influence of systemic effects of racism against Black people, which is argued to be the major contributor to disparate outcomes between Black and White patients with cancer. This review highlights evidence-based strategies that have the potential to help mitigate disparities, improve care for vulnerable populations, and build an equitable healthcare system. Lessons learned can also inform a more equitable response to other health conditions and crises.
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