As a complement to the clinical development of new anticancer molecules, innovations in therapeutic vectorization aim at solving issues related to tumor specificity and associated toxicities. Nanomedicine is a rapidly evolving field that offers various solutions to increase clinical efficacy and safety.Here are presented the recent advances for different types of nanovectors of chemical and biological nature, to identify the best suited for translational research projects. These nanovectors include different types of chemically engineered nanoparticles that now come in many different flavors of ‘smart’ drug delivery systems. Alternatives with enhanced biocompatibility and a better adaptability to new types of therapeutic molecules are the cell-derived extracellular vesicles and micro-organism-derived oncolytic viruses, virus-like particles and bacterial minicells. In the first part of the review, we describe their main physical, chemical and biological properties and their potential for personalized modifications. The second part focuses on presenting the recent literature on the use of the different families of nanovectors to deliver anticancer molecules for chemotherapy, radiotherapy, nucleic acid-based therapy, modulation of the tumor microenvironment and immunotherapy.This review will help the readers to better appreciate the complexity of available nanovectors and to identify the most fitting “type” for efficient and specific delivery of diverse anticancer therapies. Cancer causes approximately 10 million deaths per year worldwide for around 18 million new cases , 3  created by chemical engineering (e.g. nanoparticles)  or derivin vivo characterization of each of their subunits remains challenging. The second organic subfamily contains lipid-based nanoparticles that are the most represented in preclinical and clinical studies due to their unmatched biocompatibility [Chemically engineered nanoparticles form a vast class of nanovectors with a wide variety of structures, sizes and compositions , 9 Fig.. Among ttibility , 14, 15.tibility , 17. Howtibility  and hybrtibility .vβ3 integrin overexpressed on both the tumor neovasculature and some malignant cells – was also reported to improve the specific extravasation of nanoparticles in tumors [Nanoparticular vectorization is traditionally believed to take advantage of the enhanced permeability and retention (EPR) effect that results from the abnormal tumor vasculature causing preferential extravasation and increased concentration of nanoparticles in tumors , 20, 21.n tumors , 27.Overall, nanoparticles act as multimodal platforms that can be extensively engineered to improve both tumor targeting and the delivery of combined treatments to malignant cells; they are perfectly suited to increase both the half-life of therapeutic molecules in the bloodstream and their concentration in tumors while lowering their systemic toxicity . Neverthin vitro compared with untargeted liposomes [The biological world provides attractive alternatives to artificial lipid-based nanoparticles. Extracellular vesicles (EVs) are naturally occurring vesicles produced by eukaryotic cells and play important roles in intercellular communications . They naiposomes . As for iposomes . Neverthiposomes . A deriviposomes , 50. Thoiposomes –54.The trend to exploit bio-derived nanostructures for cancer therapy extends to different families of pathogens. Bacterial minicells 200 to 400 nm) are achromosomal vesicles produced by bacteria upon ectopic septation  00 nm areparvoviridae) to several hundred (e.g. herpesviridae) nanometers and they can contain a non-infectious genome composed of single- or double-stranded RNA or DNA [retroviridae) present an envelope composed of an external lipidic membrane acquired while budding from the host cell surface [Viruses are extensively studied in therapeutic vectorization due to their active cell entry mechanisms, biocompatibility and well-characterized structures. Virus-like particles (VLPs) were developed to mimic animal, plant or bacteria viruses without retaining the ability to replicate in human cells  Figs. 33 and 4. A or DNA . IcosaheA or DNA , which sA or DNA . On the  surface . As VLPs surface . Their u surface .Because of their viral nature, VLPs are perfectly adapted to the delivery of therapeutic nucleic acids  but emptContrary to VLPs for which the non-replicative nature is a major determinant of their clinical safety and intermediate immunogenicity, oncolytic viruses (OVs) display all the properties of natural viruses except that their replication is restricted to malignant cells  Fig. 4)4). The dbona fide nanovectors [To improve their intrinsic anti-cancer properties, OVs are commonly armed to vectorize therapeutic transgenes that will be expressed by infected malignant cells in the TME, thereby making them ovectors . Virusesovectors , 97. Theovectors . As withThe current standard for OV treatment is intratumoral injection with the limit that only reachable tumors can be treated, but recent evidence of viral replication in tumors following intravenous administration in patients have been reported –103. DesCancer chemotherapeutics are a large family of chemical drugs  that affChemical drugs for cancer treatment vary widely by their structures, charges and solubilities that can limit their clinical use, an illustrative example being the high hydrophobicity of taxanes . The nanin vitro to breast or prostate cancer cells, respectively, or paclitaxel to lung cancer cells after systemic administration in mice [in vivo magnetic guidance and a significant tumor mass reduction at a lower dose than with free decitabine [Other types of nanovectors are currently studied to transport and deliver chemical drugs to tumors Table . The cha in mice . Packagicitabine . Among tcitabine . Their ecitabine , 63 howeThe lack of tumor specificity for chemotherapies causes off-target effects and limits clinical efficacy by decreasing drug concentration in tumors. For instance, doxorubicin displays elevated hematological and cardiac toxicities as a free molecule . It has Current studies mostly focus on actively targeted nanodrug formulations to enhance interactions of the nanoparticles with malignant cells after having reached the TME , 24, 27.Cancer cells commonly develop resistance against chemotherapies, for instance by acquiring a multidrug resistance (MDR) phenotype. This can result from the expression of ATP-dependent transporters that promote the efflux of drugs outside the cell to escape death induction , 156. NaSome chemically engineered nanoparticle families have intrinsic physical properties that make them suitable for combined therapies. As such, gold nanoparticles can be used for photothermal therapy, which consists in a local vibrational heat generation through the absorption of specific wavelengths of light . Super PThe nanovectorization of chemotherapeutic drugs has been historically dominated by the use of organic nanoparticles Table , supportRadiosensitizationHalf the cancer patients receive radiotherapy – which exploits the low resistance of tumor cells to radiation-induced DNA damages – during their course of treatment . Overexpin vivo by click chemistry after systemic injection [2-elevating abilities or nano-radiosensitizers with diminished oxygen dependence [2 production to actively reverse hypoxia in tumors. These nanoparticles were loaded with the photosensitizer acridin orange and exhibited enhanced radiotherapy efficacy both in vitro and in vivo in a lung cancer xenograft model [Radiations not only cause direct damages to biomolecules but also generate reactive oxygen species (ROS). This phenomenon can be enhanced in tumors by the vectorization of radiosensitizing molecules that increase either ROS production in response to ionizing beams or malignant cell sensitivity to both direct and indirect radiation effects . Gold nanjection . These nnjection . The pornjection . The lacpendence . As an eft model . Hypoxiaft model , macromoft model , 187 andft model  have alsadenoviridae) naturally downregulate key proteins involved in the response to radiation-induced DNA damages [b.Internal radiotherapyThe radiosensitizer family also encompasses all molecules able to enhance tumor cell sensitivity to radiation effects by interfering with essential cellular pathways like DNA repair, apoptosis induction or cell cycle progression. As such, chemotherapeutics are used as radiosensitizers at the clinical level  and thei damages , which m damages . SiRNA-m damages . As disc damages .b.Inter2 on radiotherapy efficacy [Radionuclides have been vectorized for several years with various nanovectors like VLPs –196, EVsefficacy . In anotefficacy I-albuminefficacy . In a veefficacy I in OV-iefficacy , 205–207efficacy I and indAs for chemotherapy, the different subfamilies of nanoparticles have been massively investigated to improve the efficacy of radiotherapy, but the low biocompatibility and biodegradability of inorganic nanoparticles called for the development of alternatives. Successful delivery of radiosensitizing molecules was achieved with organic nanoparticles and bio-inspired vectors such as EVs, while engineered VLPs can be chemically coupled to radionuclides. Viruses and other bio-derived vectors are also expected to define original approaches to exploit precise biological mechanisms that are involved for instance in the cellular response to radiations.Malignant transformation results from gene alterations  that displace the equilibrium between oncogene and tumor-suppressor gene expression. These alterations can be corrected or compensated using nucleic acids (DNA or RNA) for gene editing (over-expression or knock-out), direct induction of cell death by expression of toxic genes or by modulating gene expression. As free nucleic acids are rapidly degraded in the bloodstream and do not cross cell membranes, clinical translation of cancer gene therapy requires proper vectorization . VirusesTP53. Restoring wild-type p53 functions triggers cell death specifically in highly-dividing tumor cells exhibiting genome instability. An example of a nanovector exploiting this mechanism is Gendicin, a p53-encoding adenoviral vector that was the first-in-class gene therapy treatment for head and neck cancer approved by the China Food and Drug Administration in 2003 [ING4, PTEN) in the same vector demonstrated synergistic efficacy [The most frequent genetic alterations in cancer being p53 mutations, most gene therapies consist in vectorizing a wild-type  in 2003 . While mefficacy . The enhefficacy . Regardiefficacy , 220, inNRAS oncogene demonstrated efficient gene editing in vivo along with prolonged survival in a xenograft model of rhabdomyosarcoma [EGFR in vivo specifically in xenografted lung adenocarcinoma cells [in vivo genome editing capacity [in vitro and efficiently knocked out the Catenin beta-1 gene [Other studies focus on cancer gene editing to disable key oncogenes. An oncolytic myxoma virus carrying a CRISPR cassette targeting the osarcoma . Similarma cells . Transgecapacity  and can capacity  and macrcapacity , 226 hava-1 gene . NeverthHSV-TK) gene, converting ganciclovir into ganciclovir-triphosphate and inhibiting DNA elongation [de facto expresses HSV-TK [Gene therapies for triggering specific tumor cell death include Gene-Directed Enzyme/Prodrug Therapy (GDEPT)  and cytoongation , and theongation . VLPs  have also been transported with macromolecular nanoassemblies or inorganic nanoparticles [Cellular pathways and gene expression can be precisely modulated by RNA interference (RNAi). This involves different types of small RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) that interact with specific target mRNAs and stimulate their degradation or the inhibition of their translation . The tararticles  and, morarticles .G12D-targeted siRNAs were thus shown to mediate a better inhibition of tumor growth compared to liposomes in pancreatic cancer models [in vivo to neuroblastoma cells [in vivo vectorization of siRNAs against the epigenetic regulator HDAC1 [MiRNAs are naturally transported by EVs throughout the organism to modulate gene expression in neighboring or distant cells, both in physiological and pathological conditions . This prr models ; this dir models , 259. Anma cells . Howeverma cells . Micro-oma cells ; the stuma cells , 262, whma cells , 264. Suor HDAC1  or the vor HDAC1  was alsoTo conclude, all nanovector families are investigated either in preclinical studies or clinical trials for the delivery of nucleic acids for cancer therapy Table . On the Removing life supportIn recent years, cancer treatment has rapidly evolved from directly targeting malignant cells to treating the TME as a whole , 268. ThCancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) secrete immunomodulatory cytokines, growth factors and pro-angiogenic molecules that participate in tumor maintenance , 268. A b.Reprogramming the environmentEndothelial cells are other important actors of the TME as they ensure nutrient and oxygen supply to growing tumors. To induce tumor cell death, the tumor vasculature can thus be impaired by vectorizing anti-angiogenics, mostly VEGF inhibitors or anti-VEGF siRNAs. Those have been developed as single agents over the last two decades but showed major side effects, such as hemorrhages or thromboses . Anti-anNormalizing the TME by modifying the phenotypes and functions of its cellular components has become a therapeutic strategy to beat cancer . Since rOVs display intrinsic properties (e.g. induction of immunogenic tumor cell death (ICD), release of damage- and pathogen-associated molecular patterns) that make them perfectly suited for such reprogramming approaches in cancer immunotherapy. Clinical trials reported that OV-induced ICD can be sufficient to induce an abscopal anti-cancer immune response and lead to tumor eradication , 292. OVTo vectorize immunotherapies targeting the TME , nanoparRecent clinical advances in cancer immunotherapy and TME reprogramming are yet to be enhanced efficiently by appropriate vectorization approaches. Viruses display natural abilities  for this, with OVs also exhibiting replication and oncolysis properties that can further improve their therapeutic efficacy. The development of clinical-grade viruses may be however challenging and organic nanoparticles, which are investigated in numerous preclinical studies to deliver immunomodulating proteins to tumors, offer good alternatives when considering their multiple engineering possibilities. The most efficient designs are still to be identified in clinical studies but advances in vaccination strategies using nanoparticles, for instance regarding Covid-19, may accelerate these developments. As for VLPs, EVs and bacterial minicells, their ability to vectorize biomolecules to modulate the TME has been demonstrated but clinical evidence is still missing.The last three decades have seen the discovery of a tremendous number of new anti-cancer molecules selected for their tumor-specific cytotoxicity and, more recently, for their ability to alter the TME. However, a large majority of the molecules identified on the bench fail in the clinic because of a poor efficacy/safety ratio after systemic administration. Despite personalized combinations to strike tumors on different fronts, resistance and toxicities are still major issues that limit many therapeutic applications. The advent of nanotechnologies opened an entirely novel area of research around the nanovectorization of anti-tumor therapeutics to both increase treatment efficacy and reduce associated toxicities by improving dramatically the specificity of tumor targeting. Chemically engineered nanoparticles – highly adaptable and for some relatively easy to manufacture – were the first to enter the clinic but with the current trend to improve the biocompatibility and to exploit precise biological mechanisms, bio-inspired nanovectors  are now rapidly gaining interest. These different families of nanovectors allow the vectorization of almost all anti-cancer therapeutics, including chemical drugs, radio-elements, nucleic acids, toxins and immunotherapies Table . To thisThe field of nanovectorization is overly active and has already provided important advances for cancer therapy, with clinical approvals for several simple nanoformulations Table . Current


A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology. Gene therapy as a strategy to provide therapeutic benefit includes modifying genes via disruption, correction, or replacement . Gene thex vivo retroviral delivery of a selective neomycin-resistance marker to tumor infiltrating leukocytes (TILs) extracted from advanced melanoma patients  deficiency, participated in a clinical trial which delivered a non-mutated OTC gene to the liver through a hepatic artery injection of the recombinant adenoviral vector housing the therapeutic gene. Unfortunately, Jesse passed away 4 days after treatment . The adepatients . Five papatients . Furtherleukemia . Subsequleukemia . Togethen humans .Almost two decades after these cases, gene therapy returned in clinical trials with reengineered viruses designed with safety in mind. Current clinical approaches are being scrutinized for evidence of insertional mutagenesis and adverse immunogenic reactions –18. Non-in situ. This alternative would allow the pathological mutation to be repaired while averting the risk of insertional oncogenesis. The discovery and repurposing of nucleases for programmable gene editing made this possible, beginning with the development of zinc finger nucleases (ZFN)  locus, a more attractive strategy would be to directly correct the existing genetic aberrations es (ZFN) , 21, foles (ZFN) . While tStreptococcus thermophilus, which unlike other known Cas genes, Cas9 was a large gene that encoded for a single-effector protein with nuclease activity  in the target DNA. The second component of effective targeted gene editing is a single guide RNA (sgRNA) carrying a scaffold sequence which enables its anchoring to Cas9 and a 20 base pair spacer sequence complementary to the target gene and adjacent to the PAM sequence. This sgRNA guides the CRISPR/Cas9 complex to its intended genomic location. The editing system then relies on either of two endogenous DNA repair pathways: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) . NHEJ ocA major concern for implementing CRISPR/Cas9 for gene therapy is the relatively high frequency of off-target effects (OTEs), which have been observed at a frequency of ≥50% . Currentin silico that contained both the sgRNA and the target site within the same construct. This allowed collecting Cas9 editing efficiency data in an intrinsic manner and establish a new training dataset that avoids the biases introduced through other models. Furthermore, a comparative performance evaluation to predict sgRNA efficiency of sgDesigner with 3 other commonly used tools  revealed that sgDesigner outperformed all 3 designer tools in 6 independent datasets, suggesting that sgDesigner may be a more robust and generalizable platform  is one of the most extensively used Cas9s with a relatively short canonical PAM recognition site: 5′NGG3′, where N is any nucleotide. However, SpCas9 is relatively large and difficult to package into AAV vectors (Staphylococcus aureus Cas9 (SaCas9) is a smaller ortholog that can be packaged more easily in AAV vectors but has a longer PAM sequence: 5′NNGRRT3′ or 5′NNGRR(N)3′, where R is any purine, which further narrows the window of therapeutic targeting sites. Engineered SaCas9 variants have been made, such as KKH SaCas9, which recognizes a 5′NNNRRT3′ PAM, broadening the human targeting sites by 2- to 4-fold. OTEs, however, are observed with frequencies similar to wildtype SaCas9 and need to be considered in designing any therapeutic application  can be manipulated to target RNA by providing a short oligonucleotide with a PAM sequence, known as a PAMmer ] which showed robust editing efficiency and tolerated repeated administration due to reduced immunogenic toxicity in mice immunized against AAV and Cas9  system. In this system, a catalytically inactive dead Cas9 (dCas9) is conjugated to deaminase, which can catalyze the conversion of nucleotides via deamination. For increased editing efficiency, Cas9 nickase (Cas9n) fused with deaminase is recently being more utilized over dCas9 for base editing, as the nicks created in a single strand of DNA induce higher editing efficiency. Currently, the two types of CRISPR base editors are cytidine base editors (CBEs) and adenosine base editors (ABEs). CBEs catalyze the conversion of cytidine to uridine, which becomes thymine after DNA replication. ABEs catalyze the conversion of adenosine to inosine which becomes guanine after DNA replication . Base edex vivo delivery. Electroporation is also largely used for ex vivo but can be used in vivo for certain target tissues    and T ce (HSPCs) .ex vivo gene therapy has provided therapeutic benefit for hematological disorders and cancer immunotherapy, many tissue types are not suited for this method, severely limiting its therapeutic utility for other genetic diseases. in vivo manipulation is thus needed to expand CRISPR's utility to treat a broader range of genetic diseases, such as Duchenne muscular dystrophy (DMD) . Furthermore, the cargo must reach the target tissue and bypass the vascular endothelium, which are often tightly connected by cell-cell junctions (While hy (DMD)  and herehy (DMD) . CRISPR  tissues . With sy tissues . Howeverunctions , preventunctions . In addiunctions . While aex vivo engineered with CRISPR/Cas9 to knockout the α and β chains of the endogenous T cell receptor (TCR), which recognizes a specific antigen to mediate an immune response, and the programmed cell death-1 (PD-1) protein, which attenuates immune response. The cells were then transduced with lentivirus to deliver a gene encoding a TCR specific for a NY-ESO-1 antigen, which has been shown to be highly upregulated in the relapsed tumors and thus can serve as a therapeutic target. Since then, many trials have opened for CRISPR-mediated cancer immunotherapy and is currently the most employed strategy for CRISPR gene therapy  after careful consideration of the risk to benefit ratios of this first application of CRISPR gene therapy into the clinic. During this trial, T lymphocytes were collected from the patients' blood and  therapy . A trial therapy . The modBCL11A gene (NCT03745287) as disruption of this gene increases HbF expression  to assess the long-term risks and benefits of CTX001 will dictate whether this approach can provide a novel therapeutic opportunity for a disease that otherwise has limited treatment options.The first clinical trial in the US using CRISPR to catalyze gene disruption for therapeutic benefit were for patients with sickle-cell anemia (SCD) and later β-thalassemia, by Vertex Pharmaceuticals and CRISPR Therapeutics. This therapy, named CTX001, increases fetal hemoglobin (HbF) levels, which can occupy one or two of four hemoglobin binding pockets on erythrocytes and thereby provides clinical benefit for major β-hemoglobin diseases such as SCD and β-thalassemia . The tripression –106. Genex vivo editing and subsequent therapy with modified cells, in vivo approaches have been less extensively employed. An exciting step forward with CRISPR gene therapy has been recently launched with a clinical trial using in vivo delivery of CRISPR/Cas9 for the first time in patients. While in vivo editing has been largely limited by inadequate accessibility to the target tissue, a few organs, such as the eye, are accessible. Leber congenital amaurosis (LCA) is a debilitating monogenic disease that results in childhood blindness caused by a bi-allelic loss-of-function mutation in the CEP290 gene, with no treatment options. This therapy, named EDIT-101, delivers CRISPR/Cas9 directly into the retina of LCA patients specifically with the intronic IVS26 mutation, which drives aberrant splicing resulting in a non-functional protein. The therapy uses an AAV5 vector to deliver nucleic acid instructions for Staphylococcus aureus Cas9 and two guides targeting the ends of the CEP290 locus containing the IVS26 mutation. The DSB induced by Cas9 and both guides result in either a deletion or inversion of the IVS26 intronic region, thus preventing the aberrant splicing caused by the genetic mutation and enabling subsequent translation of the functional protein  gene in human embryos, with the intention of conferring HIV-resistance, as seen by a naturally occurring CCR5Δ32 mutation in a few individuals  co-receptor , a heart condition responsible for most sudden cardiac deaths in people under the age of 30. Gene correction for this pathological mutation was achieved recently for the first time in the US in viable human embryos using the HDR-mediated CRISPR/Cas9 system. However, these embryos were edited for basic research purposes and not intended for implantation. In this study, sperm carrying the pathogenic MYBPC3 mutation and the CRISPR/Cas9 machinery as an RNP complex were microinjected into healthy donor oocytes arrested at MII, achieving 72.4% homozygous wildtype embryos as opposed to 47.4% in untreated embryos. The HDR-mediated gene correction was observed at considerably high frequencies with no detectable OTEs in selected blastomeres, likely owing to the direct microinjection delivery of the RNP complex in the early zygote. Interestingly, the maternal wildtype DNA was used preferentially for templated repair over the provided exogenous ssODN template  has led to a global pandemic . These pRecently, a CRISPR Cas12-based assay named SARS-CoV-2 DETECTR has been developed for detection of COVID-19 with a short turnaround time of about 40 min and a 95% reported accuracy. The assay involves RNA extraction followed by reverse transcription and simultaneous isothermal amplification using the RT-LAMP method. Cas12 and a guide RNA against regions of the N (nucleoprotein) gene and E (envelope) gene of SARS-CoV-2 are then targeted, which can be visualized by cleavage of a fluorescent reporter molecule. The assay also includes a laminar flow strip for a visual readout, where a single band close to where the sample was applied indicates a negative test and 2 higher bands or a single higher band would indicate cleavage of the fluorescent probe and hence positive for SARS-CoV-2 .Ruminococcus flavefaciens derived VI-D CRISPR-Cas13d variant, selected for its small size facilitating easier packaging in viral vehicles, high specificity, and strong catalytic activity in human cells. This technique was developed to simultaneously target multiple regions for RNA degradation, opening the door for a much-needed pan-coronavirus targeting strategy, given the evidence suggesting relatively high mutation and recombination rates of SARS-CoV-2  has been developed, which utilizes the RS-CoV-2 . With thThe birth of gene therapy as a therapeutic avenue began with the repurposing of viruses for transgene delivery to patients with genetic diseases. Gene therapy enjoyed an initial phase of excitement, until the recognition of immediate and delayed adverse effects resulted in death and caused a major setback. More recently, the discovery and development of CRISPR/Cas9 has re-opened a door for gene therapy and changed the way scientists can approach a genetic aberration—by fixing a non-functional gene rather than replacing it entirely, or by disrupting an aberrant pathogenic gene. CRISPR/Cas9 provides extensive opportunities for programmable gene editing and can become a powerful asset for modern medicine. However, lessons learned from traditional gene therapy should prompt greater caution in moving forward with CRISPR systems to avoid adverse events and setbacks to the development of what may be a unique clinically beneficial technology. A failure to take these lessons into account may provoke further backlash against CRISPR/Cas9 development and slow down progression toward attaining potentially curative gene editing technologies.Although CRISPR editing in humans remains a highly debated and controversial topic, a few Regulatory Affairs Certification (RAC)-reviewed and FDA-approved CRISPR gene therapy trials have opened after thorough consideration of the risk to benefit ratios. These first few approved trials, currently in Phase I/II, are only for patients with severe diseases, such as cancers or debilitating monogenic diseases. The outcomes of these trials will dictate how rapidly we consider using this system to treat less severe diseases, as the risks of the technology are better understood. A concern remains whether normalizing CRISPR/Cas9 editing for less debilitating diseases may act as a gateway for human genome editing for non-medical purposes, such as altering genes in embryos to create offspring with certain aesthetic traits. This fear of unnatural selection for unethical reasons has likely become more tangible in the public's view with the strong media attention of the edited “CRISPR babies.” The lasting effects of that trial and outcomes of the approved clinical trials will greatly influence CRISPR's future in gene therapy and begin to answer the key questions we must consider as we further explore this technology. These key questions include how to avoid the mistakes of the past, who should decide CRISPR's therapeutic future, and how the ethical boundaries of its applications should best be drawn.FU researched and drafted the article. TS and CR supervised the content. All authors wrote, reviewed, and edited the manuscript before submission.CR has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Astra Zeneca, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Loxo, and Pharmar, and is on the scientific advisory boards of Harpoon Therapeutics and Bridge Medicines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli based ALE experiments. These experiments totalled 208 independent evolutions and 2641 mutations. Additionally, mutated features were statistically associated across a total of 43 unique experimental conditions to aid in deconvoluting mutation selection pressures.Adaptive Laboratory Evolution (ALE) has emerged as an experimental approach to discover mutations that confer phenotypic functions of interest. However, the task of finding and understanding all beneficial mutations of an ALE experiment remains an open challenge for the field. To provide for better results than traditional methods of ALE mutation analysis, this work applied enrichment methods to mutations described by a multiscale annotation framework and a consolidated set of ALE experiment conditions. A total of 25,321 unique genome annotations from various sources were leveraged to describe multiple scales of mutated features in a set of 35 Escherichia coli grown on glycerol as a carbon source and one previously published with Escherichia coli tolerized to high concentrations of L-serine.Identifying potentially beneficial, or key, mutations was enhanced by seeking coding and non-coding genome features significantly enriched by mutations across multiple ALE replicates and scales of genome annotations. The median proportion of ALE experiment key mutations increased from 62%, with only small coding and non-coding features, to 71% with larger aggregate features. Understanding key mutations was enhanced by considering the functions of broader annotation types and the significantly associated conditions for key mutated features. The approaches developed here were used to find and characterize novel key mutations in two ALE experiments: one previously unpublished with .The emergent adaptive strategies represented by sets of ALE mutations became more clear upon observing the aggregation of mutated features across small to large scale genome annotations. The clarification of mutation selection pressures among the many experimental conditions also helped bring these strategies to light. This work demonstrates how multiscale genome annotation frameworks and data-driven methods can help better characterize ALE mutations, and thus help elucidate the genotype-to-phenotype relationship of the studied organism Mutations observed from ALE experiments have proven valuable for both biological discovery and applied biotechnology, such as the elucidation of the rate and mechanisms of mutation development –5 and thion data , traditiion data , 10. UltThe main concern with traditional ALE mutation analysis is the mutated genes and how the sequence changes affect their function. Identifying commonly mutated genetic features (genes or intergenic regions) across replicate ALEs, known as convergence, has been established as a primary method for identifying potentially causal mutations, or key mutations, in ALE experiments . MutatioEscherichia coli K-12 MG1655 has led to the emergence of knowledge and data resources that can help solve challenges in understanding ALE results. Genome annotation frameworks such as regulons [The accumulating wealth of information on the molecular biology of regulons , pathwayregulons , and cluregulons  describeHere, we address the challenges with finding and understanding adaptive mutations through two approaches. The first is to better identify key mutations than traditional means by seeking statistically significant mutation convergence across multiple scales of genome annotations. The second is to better understand these key mutations through their enrichment of functional annotations and their statistical associations to experimental conditions. We anticipate that the approaches described here will provide the ability to deconvolute systematic targets of adaptive mutations and their selection pressures to aid in improving ALE mutation functional analysis.E. coli K-12 MG1655 genome  for the The Sankey diagrams  visualizp-value < 0.05 (Bonferroni corrected) were considered to be significantly enriched by mutations. A mutated feature found to be significantly enriched had an asterisk (*) prepended to its label within the flow diagram . A p-value < 0.01 was used to measure significance rather than a p-value < 0.05 to reduce the amount of false-positives with associations.This dataset tracked 10 different types of experimental condition types with a total of 43 unique conditions Fig. a, d, desE. coli K-12 MG1655 based ALE experiments. We first present a small case study from a Δpgi ALE experiment [The impact of annotating mutations using multiscale genomic features was explored using 18 periment , 22 to dperiment . The secperiment  with a npgi knockout , finding that the proteins PntA and PntB were rendered non-functional through truncations [pntA gene in a replicate lacking pntA and pntB mutations. The same ALE study had seen SNPs to both sthA and its upstream regions. The mutations to these upstream regions were hypothesized to change the expression of the downstream genes in a way that benefited the Δpgi host. Advanced phenotypic characterization of the endpoint strains with pntA, pntB, sthA, and upstream mutations revealed a convergence to equivalent intracellular states, demonstrating the same phenotype achieved by different mutations. The potential impact of the upstream mutations became more evident when integrating annotations for non-coding regulatory features and observing the convergence of all small mutated features onto broader annotations such as operons . Except for promoters, the median proportions of non-coding features involved in significant convergence across ALE experiments were higher than that of coding features  in the starting strain was detrimental to population growth. The deactivation of the Rcs system through the mutations distributed across multiple operons was a key feature in the genotype which ultimately enabled the heightened aromatic amino acid levels. Though these types of mutation convergences can be manually identified with the prerequisite knowledge and detailed annotation, the growth in mutation datasets and the use of more complex organisms render their identification less likely.The convergence of small mutated features onto broader annotations and functions may not always be straightforward to manually identify with traditional methods. For example, an L-serine tolerance ALE experiment acquired adaptive mutations to plicates . The int regions . Each ofP < 0.05, permutation test, mutations to feature > 1) in ALE experiments with each broader annotation type. For example, 62% (median) of the mutated features annotated only with genomic features  were found to be significant. When considering the COGs for these small features, 71% (median) of mutated features were involved in significant convergence.It was observed that the addition of broader types of annotations to the multiscale annotation framework generally increases the number of key mutations found within ALE experiments Fig. d. This iE. coli K-12 MG1655 with some variants [The methods of top-down functional analysis and mutated feature-to-condition association analysis contextualized mutations in an ALE experiment evolving variants  on glycevariants . Besidesvariants . The repglpK, cyaA, and crr genes were the three most often mutated in this experiment, hosting 28, 21, and 10 mutations, respectively, and are within the CRP regulon  and mutations to these genes were strongly associated with the selection pressure of glycerol as a carbon source  , 30 and lon Fig. a. The CRglpFKX operon mutations. The significantly convergent glpFKX operon  hosted 30 instances of mutated features, corresponding to 28 coding SNPs in glpK, where two of these mutate a GlpR TFBS (located inside the coding sequence of glpK). These mutated features significantly enriched the GlpR regulon , which catalyzes the phosphorylation of glycerol and is a rate-limiting enzyme in glycerol metabolism. GlpK is allosterically inhibited by fructose-1,6-bisphosphate (FBP) [crr gene product, Enzyme II A (IIAGlc) [glpK have effects that could disable inhibition by FBP   and the (IIAGlc) . Two SNP(IIAGlc) . The 2 SFBP Fig. d.cyaA as there were to crr, these mutations may have accomplished similar adaptive effects. The presence of mutations to either of these targets was inversely correlated with the other . A cAMP molecule will bind with CRP and promote the transcription of CCR genes involved in secondary carbon source metabolism, including the glpFKX operon. Before cAMP can do so, this functionality of AC must be activated by binding with a phosphorylated IIAGlc. In the presence of phosphotransferase system (PTS) sugars such as glucose, IIAGlc will instead bind and inhibit GlpK. In the absence of PTS sugars, IIAGlc will become phosphorylated, bind, and activate AC’s ability to produce cAMP, ultimately reducing the effect of CCR [Though there were approximately twice as many mutations in her Fig. c, indicat of CCR .cyaA gene had a disruptive effect. cyaA hosted a total of 21 unique mutations. The mutations that affect cyaA were of different types: SNPs, deletions, and mobile element insertions (MOB). One-third of the mutations to cyaA, the two MOBs and five frameshifting deletions, had disruptive effects on the coding sequence , which both describe the regulation of the PTS system, a key contributor to the CCR system. All mutations to crr landed in the PTS EIIA type-1 domain, which hosts the IIAGlc phosphorylation site. 70% of the mutations to crr were predicted to have a structurally destabilizing change on their host structure . Additional evidence towards the mutations having a disruptive effect on crr is the possible epistatic relationship between cyaA and crr mutations along with the mutations to cyaA having the clear potential to disrupt its functionality. This evidence suggested the potential non-essentiality of IIAGlc in this evolution.The evidence of potential mutation effects to P < 0.001, permutation test, mutations to feature > 1) and significantly associated with the designed selection pressure of growth on glycerol . The functions of these regulons contextualized their mutated genes, further informing on the biological systems potentially targeted by adaptation. Further investigation into the mutated genes informed on the possible beneficial mechanistic effects of their mutations. The mutations to glpK suggest the increase in its reaction rate through the disruption of an inhibition mechanism. The mutations to cyaA and crr suggest the disruption of cAMP synthesis, resulting in CCR maintenance in the presence of a carbon source that would normally dampen CCR. Such induction of CCR with mutations resulting from an experimental evolution on glycerol has been previously observed [glpK, cyaA, and crr had the strongest and most interpretable signals of adaptation.This ALE experiment generated 148 mutations, an amount prohibitive to comprehensive manual functional analysis. The methods of top-down functional analysis and selection pressure associations generated valuable evidence that revealed a potential solution for increasing glycerol metabolism while maintaining the repression of metabolic systems for other secondary carbon sources. Of the many mutated regulons for this ALE experiment, three were emphasized by being both significantly converged upon by mutations . Collectively, 27 mutations unique to endpoints were analyzed for this ALE study. The original Mundhada et al. study [thrA, lrp, rho, argP, pykF, and eno contributed to L-serine tolerance and fitness in minimal-media.The methods of top-down functional analysis and mutated feature-to-condition association analysis contextualized mutations in an ALE experiment tolerizing a genetically engineered L-serine , resultil. study  revealedP = 0.0013, permutation test, mutations to feature > 1) by mutated features  except rhoL-rho were significantly associated  with L-serine tolerance and starting strain mutations  experienced a 1 bp DEL in two endpoints , where another endpoint hosted a 1 bp DEL in an activating GcvA TFBS . lrp is a global regulator for the leucine response system and has experimental evidence demonstrating its participation in L-serine tolerance [lrp and rho coding sequence mutations were found to contribute the most fitness among all endpoint mutations of this experiment [The operons for global regulators olerance . The rolperiment .This ALE experiment generated a variety of mutations to both coding and non-coding regions, where mutations to non-coding features not considered in the original study provided evidence for new key mutations. The methods of top-down functional analysis and selection pressure associations generated valuable evidence that revealed a potential optimization for the strain’s glycine auxotrophy. The richer annotations for non-coding features on the genome revealed the high frequency of regulatory features targeted by ALE mutations and facilitated the discovery of previously unrecognized mutated regulatory features. Significant enrichment of features on the multiscale annotation framework emphasized the systems in which the ALE mutations affected, informing on the normally more elusive non-coding mutations. This emphasis on higher-level features revealed previously unconsidered changes to the glycine cleavage system that could benefit the host strain. These results serve to promote the value of the evidence generated by the methods of this work in finding and understanding key mutations.To better identify and understand causal mutations from ALE experiments, this study developed a multiscale genome annotation framework and applied a statistical enrichment method for mutated features across annotation scales. Using this framework, it was found that (1) ALE mutations target a variety of regulatory features including promoters, attenuator-terminators, and ribosomal binding sites, (2) mutated non-coding regulatory features were often involved in significant convergence events, and (3) the method of bottom-up convergence from small to large features on the multiscale annotation framework found more key mutations than when considering only genes and intergenic regions for mutation convergence. The convergence of mutated features onto broader functional annotations additionally enabled a top-down approach to mutation functional analysis, where one can first consider biological functions hosting mutations before investigating the numerous smaller mutated features. Further, we computed statistical associations between a large set of experimental evolution conditions and mutated features. These associations provided evidence towards clarifying the selection pressures for mutated features among the multiple conditions describing each ALE experiment. Taken together, the results presented here have several implications.lrp and rho genes, was made explicit with the inclusion of regulatory features in mutation annotations. These approaches can therefore lessen the challenge of finding key mutations by providing enhancements to traditional methods.First, the identification of key mutations was enhanced by seeking mutations involved in statistically significant convergence events across multiple scales of genome annotation. By including regulatory feature annotations alongside genes, a larger proportion of an experiment’s mutations were placed in features with known functions, which were subsequently connected to broader annotation types such as operons and functional annotations. Being able to connect more mutations to known features resulted in more convergence events across the annotation framework. Additionally, the application of a statistical enrichment test can be used to prioritize convergence events in the followup work of mutation functional analysis. This will become more important as ALE experiments increase in scale. All mutated regulatory feature types were involved in statistically significant convergence events, and except for promoters, had a higher median proportion of their mutations involved in these events than mutated genes. These results suggest that mutations to non-coding regulatory features should be considered as important as coding features when searching for key mutations, though standard genome annotations often lack non-coding regulatory features. When only considering mutations from significantly converged upon features, the amount of significant or key mutations on larger-scale annotation types  remains larger than those on smaller annotation types . The convergence of a variety of small mutated features onto broader annotations also serves to maximize the amount of evidence used in identifying the overall changes of an adaptive genotype. In its application, this method identified new regulatory key mutations for a published ALE study on L-serine tolerance , demonstSecond, the functional analysis of key mutations was enhanced by considering the functions of the features upon which mutations converged and the conditions that mutated features were associated with. We found that a top-down mutation functional analysis proved valuable in contextualizing key mutations according to the high-level functional annotations to which they were connected and shared with other key mutations. The three most frequently mutated genes of strains from the ALE experiment on glycerol  all convE. coli indel mutation rates have been observed to be higher in mononucleotide runs of 4 or more [E. coli K-12 MG1655. To use these methods with other organisms would require knowledge bases that describe the genes and regulatory architecture of those organisms’ genome, along with the biological functions that the genes contribute to. Evolution experiments often also include midpoint or intermediate samples. The results of this work only included endpoint samples, as not all experiments had midpoints, to enable a uniform analysis method across all experiments. The inclusion of midpoint samples could provide further evidence of mutation convergence through the dynamics of clonal interference, as well as the opportunity for mutation time series analysis. Correlations between mutated features proved useful for interpreting the relationship between the mutated cyaA and crr genes from the glycerol carbon source ALE case study. Applying the same correlation method on a larger scale mutated feature set tends to generate less interpretable results, though statistical evidence of relationships between mutated features would still be valuable. More sophisticated methods for establishing these relationships within large-scale sets of mutated features are necessary to extract meaningful interpretations. Finally, the key mutations found in this work were derived from sets of mutations of a scale that still can be managed manually, albeit with great effort. As ALE datasets grow and automation-enabled studies increase in overall sample number, this work’s methods will become necessary to comprehensively consider and understand an ALE experiment’s mutations.The methods presented here demonstrate the value in leveraging the diverse available resources describing ALE variants, but are not without limitations. The procedure for finding the statistical significance of mutation convergence assumes that each nucleotide on the genome has the same probability of being spontaneously mutated, leading to features having the probability of being mutated proportional to their length in nucleotides. Nucleotides can have varying mutation rates; for example, wild type  or more . Paramet or more , 45, gen or more , and gen or more , 48. TheHere, we have reported on data-driven approaches to better find and understand ALE key mutations. We demonstrated how a multiscale genome annotation framework and statistical association methods can better identify and characterize adaptive mutations generated through controlled evolution experiments. We anticipate that this workflow will be leveraged in the future to provide deeper insights into the vast amounts of -omics data that are generated for targeted microorganisms amenable to ALE and provide the blueprints for similar data-driven annotation frameworks.E. coli K-12 MG1655 or derivative thereof, due to the availability of experimental data and genome annotations. Thirty five ALE experiments, each using a unique set of conditions and selection pressures, were exported from ALEdb [This work focuses on ALE experiments with a starting strain of om ALEdb , 49–57. Only mutations from endpoint flask samples  were considered for the ALE experiments used in this work. Both clonal and population samples were included in this work. If the same mutation was available in more than one sample from an endpoint flask, the instance of the mutation with the highest sample frequency was chosen to represent all instances of the mutation from the flask.The ALE experiment conditions metadata used in the associations was gathered from the metadata reports available from ALEdb  for eachMatplotlib version 3.0.3 [seaborn version 0.9 Python software packages [Unless otherwise stated, figure plots were generated using on 3.0.3  and seabpackages .Floweaver Python software package [The mutation flow diagrams of Figs.  package .muts-needle-plot Javascript software package [The mutation needle plot of Fig.  package .breseq pipeline version 0.33.1 [E. coli K-12 MG1655 reference genome. The reference genome was either the NCBI accession NC_000913 version 3 reference genome or a derivative based on this NCBI genome [The n 0.33.1  was usedI genome . DNA-seqI genome .mutfunc web application [The predicted disrupted effects of mutations to the genes in the glycerol evolution results were generated using the lication .Software scripts and data essential to the publication is available through a github repository .Additional file 1: Supplementary figures and tables.


Significance: Tumor detection and margin delineation are essential for successful tumor resection. However, postsurgical positive margin rates remain high for many cancers. Raman spectroscopy has shown promise as a highly accurate clinical spectroscopic diagnostic modality, but its margin delineation capabilities are severely limited by the need for pointwise application.Aim: We aim to extend Raman spectroscopic diagnostics and develop a multimodal computer vision-based diagnostic system capable of both the detection and identification of suspicious lesions and the precise delineation of disease margins.Approach: We first apply visual tracking of a Raman spectroscopic probe to achieve real-time tumor margin delineation. We then combine this system with protoporphyrin IX fluorescence imaging to achieve fluorescence-guided Raman spectroscopic margin delineation.Results: Our system enables real-time Raman spectroscopic tumor margin delineation for both ex vivo human tumor biopsies and an in vivo tumor xenograft mouse model. We then further demonstrate that the addition of protoporphyrin IX fluorescence imaging enables fluorescence-guided Raman spectroscopic margin delineation in a tissue phantom model.Conclusions: Our image-guided Raman spectroscopic probe-tracking system enables tumor margin delineation and is compatible with both white light and fluorescence image guidance, demonstrating the potential for our system to be developed toward clinical tumor resection surgeries. Due to the infrequency of Raman scattering events, which necessitates relatively long acquisition times  of the Raman spectroscopic probe as well as the diagnostic spectroscopic information for each measurement acquisition. Together, the data are overlaid onto imaging of the surgical FOV to provide an augmented reality (AR) display of the FOV. We demonstrate our system is capable of accurate lesion mapping of both 22.1,ex vivo margin delineation setting. Computed coordinate locations for each of the fiducial markers and the probe tip were compared to ground truth coordinates, generated as the mean  tumor biopsy samples and four human skin normal biopsy samples were obtained from the Imperial College Healthcare Tissue Bank (ICHTB) see Sec. . Ex vivo2.42.5in vivo and 80 spectra from the SW1222 xenograft tumors in vivo at 12 days after implantation. Spectra were cropped to between 600 and In vivo image-guided Raman spectroscopic margin delineation of a SW1222 xenograft tumor in a third mouse was then performed using the image-guided Raman spectroscopic probe-tracking system with a 785-nm laser at 100 mW power output and a 1-s integration time through prospective application of the previously developed PLS-DA model.Two female nu/nu mice  were subcutaneously injected with 2.6MDA-MB-231 cells (ATCC) for optical tissue phantoms were grown at 37°C and 5% 2.7ex vivo chicken muscle tissue, raw ex vivo chicken fat tissue, and the 0-, 2, 4, and Ex vivo fluorescence-guided Raman spectroscopic margin delineation was performed on additional raw chicken tissue specimens with 0, 2, or Using the image-guided Raman spectroscopic probe-tracking system, 25 to 30 Raman spectra were collected from raw 2.8Human samples used in this research project were obtained from the ICHTB. ICHTB is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. ICHTB is approved by Wales REC3 to release human material for research (17/WA/0161), and the samples for this project (R19022) were issued from the ICHTB Collection.All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Guidance for the Operation of Animals (Scientific Procedures) Act 1986  and United Kingdom Coordinating Committee on Cancer Research Guidelines for the Welfare and Use of Animals in Cancer Research.33.1a priori knowledge of probe geometry to determine the position and orientation (pose) of the probe for spatial diagnostics .Our image-guided Raman spectroscopic probe-tracking system combines widefield imaging with Raman spectroscopic information and consists of a camera for white light/fluorescence imaging, a handheld fiber-optic probe, a laser and spectrograph for Raman spectroscopy, an excitation light source, collection filter optics for fluorescence imaging, and a computer with integrated software for clinical control . ClinicaSupplemental Materials), which is then combined with a priori knowledge of the probe and marker geometry for ratiometric calculation of the pose of both the spectroscopic probe and the probe tip (point of measurement acquisition) . The HSV color space is used here as it has been shown to be superior to the RGB color space for surgical tool tracking due to its decoupling of the chromaticity and luminance components.,Supplemental Materials) overlays the tracked location of the probe tip onto the clinical imaging video and records Raman spectral signal in real time. Upon user identification of a region of interest, the system  records both the location of the probe tip and a Raman spectral signal for diagnosis. This Raman spectral signal is then diagnosed in real-time through application of a previously developed diagnostic model   and the diagnoses displayed at the probe tip coordinates are overlaid onto the clinical imaging video. Positive diagnoses are then connected to form a boundary that outlines the lesion margin. Importantly, the algorithm operates independently on each input video frame, accessing any previously stored diagnostic measurements to update margin delineation at each step, to ensure robust performance following temporary occlusion of the spectroscopic probe .In the probe-tracking schema we developed, initial manual identification of the colored, fiducial probe markers in the FOV enables HSV -based image segmentation  within a clinician-facing graphical user interface (GUI) that provides functionality for system calibration, diagnostic spectroscopic model development, and margin delineation. The image-guided Raman spectroscopic probe-tracking system control software provides both a raw video input display and an AR display of the surgical FoV, with spatially co-registered spectroscopic diagnostic information overlaid onto the raw video input.For Supplemental Materials). Validation of our image-guided Raman spectroscopic probe-tracking system was performed using fresh chicken tissue, discriminating between chicken muscle and chicken fat tissues. Here chicken tissue served as a good model for validation of probe tracking accuracy, with strong visual and spectroscopic lipid contrast enabling ready visual and Raman spectroscopic tissue discrimination. This enabled evaluation of probe tracking accuracy independent of Raman spectroscopic diagnostic accuracy. Our probe tracking system achieved a mean probe tip tracking error of –Supplemental Materials]. Similarly, computational performance, measured during a mock spatial diagnostic procedure, yielded processing times of Supplemental Materials].To examine the potential for clinical operation of our system, we first investigated the probe tracking and margin delineation accuracies as well as the computational performance of the system . These features include adjustable safety margins, automatic suggested measurement locations, adjustable diagnostic thresholds, and video stabilization. Each of these features is intended to maximize robustness of our system to different clinical settings and maintain clinician diagnostic control.To further support clinical operation, we implemented several features designed to enable users to tailor spatial spectroscopic diagnostics to a particular patient or lesion , we evaluated the margin delineation accuracy of our image-guided Raman spectroscopic probe-tracking system for three ex vivo chicken tissue specimens. Delineated areas closely resembled the ground-truth areas in each case, as determined via manual image segmentation . Balancing true positive delineated area with false negative and false positive delineated areas, the best results were obtained using a safety margin of 1.5 mm for this particular setup, with a mean delineated true positive area of 86.5%, false negative of 13.5%, and false positive of 21.7% .Using our previously developed PLS-DA model . Using this model, we were able to effectively delineate a region of cancerous tissue from surrounding normal (muscle/non-cancerous) and normal (fatty) tissue for a human SCC biopsy sample, confirmed histologically using adjacent H&E stained sections . The system displays real-time spectroscopic information and automated PLS-DA diagnosis for each acquisition, providing an AR display of the specimen where the locations of negative (non-cancerous) acquisitions are displayed as green squares (with numbers indicating order of acquisition) and the locations of positive (cancerous) acquisitions are displayed as red squares . However, our image-guided Raman spectroscopic probe-tracking system significantly outperformed fluorescence imaging alone for the We next developed a PLS-DA model to discriminate chicken muscle tissue from a non-fluorescent  . StudiesThis result thus demonstrates the complementarity of fluorescence imaging with Raman spectroscopic diagnostics, whereby fluorescence imaging enables rapid identification of fluorescence positive regions followed by spatial spectroscopic diagnosis for accurate margin delineation independent of variations in fluorescence intensity. As such, by developing diagnostic models that combine both fluorescence imaging information and Raman spectroscopic information, the two modalities could yield more accurate margin delineation in combination than either modality would achieve in isolation. However, an important caveat to note is that margin delineation accuracy via our image-guided Raman spectroscopic probe-tracking system is dependent on both the probe tracking accuracy and the accuracy of the underlying diagnostic model for spectroscopic discrimination between tumor and healthy tissue. Given the significant spectral differences between the optical tissue phantoms and the chicken tissue used for these experiments, the results presented here reflect an idealized case where the underlying diagnostic model has a 100% discrimination accuracy.4,,,High information content optical spectroscopies such as Raman spectroscopy have long held promise for tumor margin delineation. However, the long acquisition times required have thus far restricted intraoperative Raman spectroscopy systems to point-based applications that do not provide clinicians with a visual demarcation of a tumor boundary.ex vivo and in vivo settings.Our image-guided Raman spectroscopic probe-tracking system is designed to bridge the gap between the high molecular information content of Raman spectroscopy and the visual-spatial information provided by white light/fluorescence imaging. Through tracking the pose of a Raman spectroscopic probe during spectral acquisitions, our system enables an AR display of the surgical FOV, overlaying spatially co-registered spectroscopic diagnoses onto white light/fluorescence video image input for margin delineation. This combination of spatial and spectroscopic data thereby achieves rapid and accurate spectroscopic tumor margin delineation for both ,in vivo. Importantly, probe tracking accuracy and safety margin determination are both highly dependent on the working distance between the camera and the sample as well as any magnification optics in use. As such, optimization of these parameters is essential for robust clinical implementation. Finally, for fluorescence-guided approaches, care will need to be taken to develop diagnostic models that include data from tumors with varying fluorescence intensities in order to maximize performance.in vivo and ex vivo.Although our approach to tumor margin delineation has potential, several limitations will need to be overcome in order to enable successful clinical application. First, performance should be enhanced to enable real-time operation. Although much of this enhancement could be achieved through improved code parallelization and computing hardware, this is also likely to require the use of a more sensitive Raman spectrometer in order to reduce signal acquisition times. Second, while the system currently provides a 2D AR overlay of spectroscopic and visual information, robust clinical application will likely necessitate the use of multiple cameras for stereo vision.In conclusion, the combination of Raman spectroscopic diagnosis with fluorescence imaging and computer vision probe tracking thus opens exciting opportunities for clinical tumor resection guidance. Given the improved cancer detection sensitivity spectroscopic methods have consistently shown relative to white light/fluorescence imaging modalities, our image-guided Raman spectroscopic probe-tracking system will likely enable more accurate tumor margin delineation and could lead to improved rates of complete resection and thus better patient outcomes.Click here for additional data file.


PurposeCanada has the second-highest opioid use in the world. Despite knee and shoulder arthroscopy being among the most commonly performed orthopaedic procedures, there exists little guidelines for pain management.MethodsA survey was developed and distributed to members of the Arthroscopy Association of Canada. The objectives were: to understand opioid prescribing patterns after knee and shoulder arthroscopy, to determine if surgeons believe opioid over-prescription is an issue and to identify other pain management strategies surgeons are regularly using.ResultsA total of 38 responses were included (38.3%). Eighty-two percent of surgeons felt opioid over-prescription was an issue in arthroscopic surgery. The average post-operative knee or shoulder arthroscopy prescription included a total of 156 +/- 84.4 (0-400) mg of oral morphine equivalents (OMEs). Less than one-third of respondents (29%) had received formal peri-operative pain management training. Fifty-five percent of respondents felt that non-opioid medications do not provide adequate pain relief after arthroscopic surgery. Nearly all respondents (95%) stated they would change their prescription practice if high-quality evidence were to suggest that they should do so.ConclusionsThe majority of respondents identified opioid over-prescription as a problem after arthroscopic surgery. Surgeons are prescribing five times the amount of OMEs to patients that previous literature suggests the median patient uses after arthroscopic knee surgery. Surgeons generally state they would reduce or eliminate opioid prescriptions to arthroscopy patients if high-level evidence were to emerge suggesting that adequate pain control could be achieved without the use of narcotics. Canada has the second highest per-capita opioid use in the world, with a threefold increase in the rate of prescription opioid use over the past ten years -2. The GOrthopaedic surgeons prescribe more opioid medications, including long-acting narcotics, than any other surgical specialty -7. A recIn 2006 knee and shoulder arthroscopy were found to be among the most commonly performed orthopaedic surgery procedures . DespiteQuestionnaire developmentWe assembled a focus group consisting of an author with statistical analysis experience and orthopaedic surgeons who perform knee and shoulder arthroscopy regularly. An initial draft of the questionnaire was made by one of the authors (SE) and then distributed sequentially to the focus group consisting of two orthopaedic surgery residents, a statistician, and a fellowship trained arthroscopic surgeon for feedback on the content, clarity and comprehensiveness of the survey. Questions were developed to examine respondents’ demographic characteristics, current opioid prescribing patterns after knee and shoulder arthroscopy, non-opioid pain management strategies surgeons are currently utilizing after knee and shoulder arthroscopy and surgeons’ perceptions of the opioid crisis in Canada. The final questionnaire, which had approval from the members of the initial focus group, consisted of 23 questions using multiple choice, Likert scales, limited commentary, and open responses. A complete version of the survey can be found in Appendix 1. This study received ethics approval from the Hamilton Integrated Research Ethics Board (REB No. 7560) on July 18, 2019.Questionnaire administrationThe survey was distributed to all physician members of the Arthroscopy Association of Canada (AAC) electronically through SurveyMonkey (N = 99). The survey was initially distributed on July 23, 2019, and reminder e-mails were sent on August 14, 2019 and September 6, 2019. Responses for the survey were closed on September 13, 2019. Responses were restricted to a single response per individual. Individuals who stated that they did not perform arthroscopic knee or shoulder surgery were excluded from the study. After the end date of the survey, the data from SurveyMonkey was exported to a secure Excel spreadsheet  prior to data analysis.Statistical analysisThe statistical analysis plan was established a priori and contained within the REB submission. All responses were reported in descriptive statistics, using proportions or mean ± standard deviation (SD) as appropriate. Medication doses were converted into oral morphine equivalents (OMEs) using the Oregon Pain Guidance online calculator tool . Some LiA total of 39 surveys were completed (39.4% response rate). Of these surveys, one participant indicated that they do not perform knee or shoulder arthroscopy and therefore was excluded. Thus, 38 responses were included in the final analysis. Responses to each of the survey questions can be found in Appendix 2.Characteristics of the respondentsAll respondents were physician members of the AAC and were fully licensed orthopaedic surgeons in independent practice. All ten Canadian provinces were represented, with Alberta (29%) and Ontario (24%) being most common Table . PrimaryPeri-operative pain management strategiesMost respondents routinely utilized cryotherapy after knee (79%) and shoulder (68%) arthroscopy. Over half of all respondents also performed an intra-articular injection at the conclusion of their knee arthroscopy cases (55%), compared to 40% who did so for the shoulder. Routine nerve blocks were more common in shoulder arthroscopy (42%) compared to knee arthroscopy (29%).Practice patternsThe respondents reported that on average, 88% (standard deviation = 23%) of their post-arthroscopy patients received a prescription for an opioid medication. Tylenol #3 was the most commonly prescribed first-line opioid (31%), followed by hydromorphone (25%) and tramadol (31%)  and acetaminophen concurrently with any opioid medication Figure . Two-thiPeri-operative pain management trainingLess than one-third of all respondents (29%) had received formal peri-operative pain management training. Among those who had received such training, 91% said it had “some” or “a great deal” of influence on their prescription patterns. As well, 79% of those who had not received any formal training felt that such training would be useful to them.Attitudes regarding opioid prescription in orthopaedicsNearly all 92%) respondents agreed that opioid over-prescription is a problem in surgery generally, and orthopaedics specifically  and 88% percent of patients had a surplus of unused opioids . Our stuPost-operative opioid prescriptions represent only one of several ways to manage patients’ post-operative pain. There are many studies supporting the use of pain control adjuncts such as intra-articular injections, nerve blocks, and cryotherapy after knee and shoulder arthroscopy -20. Our Approximately 60% of surgeons responded to the survey stating that they routinely discussed the risks of opioid use with their patients prior to prescribing them. Hartford et al. recently published their study where patients were prospectively enrolled after either open hernia repair or laparoscopic cholecystectomy and were either treated with standard pain management strategies or with a multimodal intra- and postoperative analgesic bundle, including promoting co-analgesia, opioid-reduced prescriptions, and patient education . They foThe main strength of this study was the fact that the survey received responses from surgeons of a variety of practice types including academic and community surgeons across Canada. Furthermore, the survey was designed and piloted by a test group of orthopaedic surgeons. Limitations of this survey include the possible variability amongst the amount of post-operative pain different arthroscopic knee and shoulder procedures cause. For instance, one study showed that patients treated with a meniscal repair required significantly more opioids post-operatively than the average knee arthroscopy patient . FurtherMost Canadian surgeons state that opioid over-prescription in arthroscopic surgery is an issue, however, there is clinical equipoise amongst respondents as to whether non-opioid prescriptions would provide adequate pain relief to patients after elective knee and shoulder surgery. Furthermore, surgeons indicated that they are willing to change their opioid-prescribing practice if strong evidence suggests emerges to suggest that they are not necessary. Therefore, high-quality studies are needed to determine how much, if any, opioids patients actually require to manage post-operative pain after elective knee and shoulder arthroscopy. One respondent in our survey remarked that official AAC guidelines on opioid prescribing would be useful. Future high-quality studies should be used to form evidence-based guidelines surrounding opioid prescriptions after elective knee and shoulder arthroscopy.The majority of respondents identified opioid over prescription as a problem after arthroscopic surgery. Surgeons are prescribing five times the amount of OMEs to patients that previous literature suggests the median patient uses after arthroscopic knee surgery. Surgeons generally state they would reduce or eliminate opioid prescriptions to arthroscopy patients if high level evidence were to emerge suggesting that adequate pain control could be achieved without the use of narcotics.


We are witnessing a tremendous outbreak of a novel coronavirus (SARS-CoV2) across the globe. Upon exposure to different population and changing environment, the viral strain might experience different mutational bias that leads to genetic diversity among the viral population. Also, the diversification can be influenced by distinct selection pressure on different viral genomes. We have carried out a comparative genomic analysis of 82 SARS-CoV2 genomes. We have evaluated their evolutionary divergence, substitution pattern, and rates. Viral genomes under distinct selection pressure have been identified. Sites that experience strong selection pressure also have been identified. Our result shows that the translational preference of a few codons is strongly correlated with the mutational bias imposed by genome compositional constraint and influenced by natural selection. Few genomes are evolving with a higher mutational rate with a distinct signature of nucleotide substitution in comparison to others. Four viral strains are under the effect of purifying selection, while nine SARS-CoV2 genomes are under strong positive selection bias. Site analysis indicates a strong positive selection pressure on two codon positions at 3606th and 8439th positions. Our study elucidates adaptation of few SARS-CoV2 viral strain during the outbreak shaping by natural selection and genomic compositional constraints. S gene, which encodes spike glycoprotein, other genes that encode for the envelope E protein and the membrane protein M, region encoding the nucleocapsid N protein followed by 3'-UTR [Recent pandemic of a new coronavirus, SARS-CoV2, infects more than 40 million people and we have witnessed 1 million deaths already. Genomic comparison of SARS-CoV2 from infected individual shows 79.6% sequence identity to SARS-CoV and 96% identity to bat coronavirus, BatCoV RaTG13, which suggests a possible bat origin of the new virus [y 3'-UTR . Data avy 3'-UTR . Thus, iHere, we have carried out a detailed comprehensive analysis of the coding region of 82 SARS-CoV2 genomes from diverse population across the world to understand their acquired genetic diversity. We have evaluated their evolutionary divergence, substitution pattern, and rates. Also, we have identified the viral genomes among the population which is under evolutionary selection pressure. The sites of the genome that experience particular selection pressure across the micropopulations also have been identified.NCBI Genome database contains full-length genomic sequences of 92 SARS-CoV2 viral strains from the human host across the world. Analysis of the annotation of genome and initial alignment revealed 10 genomes with the missing region. Those genomes were discarded from further analysis. The final set retained 82 viral genomes and the details of the genomes are shown in Table The whole-genome alignment of 82 SARS-CoV2 genomes was performed with Clustal Omega . AlignedStatistical quantities like nucleotide frequencies, codon usage pattern, and transition/transverse rate were calculated using MEGA-X. Sequences were then analysed for the identification of the best-suited nucleotide substitution model by performing Maximum Likelihood fits on 24 different substitution models. The Tamura-Nei model with nonuniformity of evolutionary rates among sites modelled by using a discrete Gamma distribution (+G) with 5 rate categories was found to be the best-suited one, judged by the lowest Bayesian Information Criterion score. The estimated gamma shape parameter was found to be 0.05. Estimation of evolutionary distance between sequences was calculated by calculating the number of base substitutions per site between sequences using the maximum composite likelihood model considering the rate variation . PatternThe phylogenetic tree was constructed by using MEGA-X . The trep values, were considered separately to identify site-wise selection pressure throughout the genome using the Datamonkey web-server (https://www.datamonkey.org/). Genomes with evidence of purifying and positive selection were aligned separately to obtain in-frame codon alignment using MAFFT, and then the phylogenetic trees were reconstituted using the maximum likelihood method. The aligned sequences with the reconstituted phylogenetic trees were then analysed with the SLAC (single likelihood ancestor counting) method to reconstruct the ancestral state using the maximum likelihood method [A codon-based selection pressure analysis was performed to identify purifying selection (dN < dS) and positive selection (dN > dS) on the CDS of 82 genomes using the Nei-Gojobori method  implemend method .SARS-CoV2 genomes are found to be AT/U-rich, as observed in other related RNA viruses. The average AT/U content is 62.07 ± 0.004% . HoweverGenomes of (-)-RNA viruses are more AT-rich than (+)-RNA. Particularly, compositional bias towards high A-content was previously reported in the coding strands for (-)-RNA . Among tCodon usage pattern also reveals more frequent utilization of codons with U at the first codon site. For example, leucine is encoded by six codons; among them, two codons, UUA and UUG, are more frequently utilized with RSCU values of 1.35. Among the rest of the four codons with C at the first site, CUU is associated with RSCU values of 1.25, while the other three codons are less frequently used . It has been often observed that both translational selection and compositional constraints dictate the codon utilization variation. Overall, we have observed that the compositional constraints mostly dictate the translational biases for SARS-CoV2 genomes. Apparent from the RSCU values of the codons in different SARS-CoV2, genomes show the clear translational preference of few codons. Notably, selection pressure also induces the nonuniformity of codon usage under the influence of compositional bias.We have identified 119 variable regions in 82 SARS-CoV2 genomes. Among them, 28 are the parsimony-informative sites where at least two different nucleotides appear at least in a frequency of two. These changes lead to 79 variable sites in the translated proteins; among them, 17 are the parsimony-informative sites. We have further assessed the evolutionary divergence in 82 genomes by calculating the number of base substitutions per site between a pair of genomes using maximum composite likelihood model, where the rate variation among sites was modelled using a gamma distribution with the shape parameter of 0.05.Results are summarized in We have further analysed the genetic diversity of SARS-CoV2 genomes during the pandemic using phylogenetic analysis using the maximum likelihood method. Viral genomes from the USA are often closely clubbed with the viral genomes from China. Notably, the first SARS-CoV2 outbreak was reported in Wuhan, China. Thus, the Chinese viral strain is the probable ancestral origin of the SARS-CoV2 genomes reported from the USA. Interestingly, two Indian viral genomes from the same state are clubbed differently suggesting different ancestral origins. Genome with accession number MT012098.1 clubbed together with genomes from USA and China while the other genome (accession number: MT050493.1) is closely related to the genome from Taiwan, China (accession number: MT192759.1).Noteworthy, viral genomes from Brazil, Australia, South Korea, Sweden, and Italy are clubbed together in a distinct subclade with a viral genome from California, USA. This clade does not contain any viral genome from Chinese origin which indicates that this group of SARS-CoV2 virus is evolved during the later stages of a pandemic. These genomes are characterized by high and distinct mutational biases thus possibly evolving with selection pressure.Our analysis clearly shows that the genetic variations in SARS-CoV2 genomes are not evenly distributed. We have therefore analysed all the 82 genomes to identify those genomes evolving with either positive or negative selection bias. We have performed a codon-based test to identify selection pressure in genomes using the Nei-Gojobori method. The probability of rejecting the null hypothesis of strict-neutrality (dN = dS) in favour of the alternative hypothesis (dN < dS for purifying selection and dN > dS for positive selection) is shown in We have further analysed the genomes with positive and negative selection bias separately for the identifications of sites under strong selection biases. This segregation process helps us to identify strongly selected sites in those viral strains evolving under strong overall selection pressure and distinct mutational bias compared to the rest of the circulating viral strains. During the early pandemic period, the rate of mutations is high which is strongly influenced by the genetic diversity of the exposed host population. The synonymous mutations are much higher than the nonsynonymous mutations. All the mutations are not significant, and mutations of the naturally selected viral strains only persist. Thus, the genome segregation based on the overall selection pressure ensures the identification of significantly selected sites. In the two datasets, we have included the viral genome from Wuhan, China, as a reference genome of origin and another sequence from USA  which shows a higher degree of evolutionary divergence . The SLASARS-CoV2 genomes with purifying selection evolved with strong biases, as the calculated dN/dS of 0.42 which is much lower than 1. Site analysis clearly shows that more sites under strong negative selection bias in comparison to the number of sites with positive selection bias . Three cHere, we have carried out a detailed evolutionary analysis on 82 SARS-CoV2 genomes obtained from different demographic regions of the world. We have observed that even in this short evolutionary timescale, there is emerging genetic diversity across the viral population. Particularly, few genomes are evolving with a higher mutational rate with a distinct signature of nucleotide substitution. We found four viral genomes are under the effect of purifying selection, while nine SARS-CoV2 genomes that include genomes from Brazil, Australia, India, and the USA are under strong positive selection bias. Site analysis indicates that two sites at 3606 and 8439 on 3C-like proteinase and spike protein, respectively, are evolving under strong positive selection bias where a hydrophobic amino acid changes to phenylalanine which makes these sites capable of interacting strongly with interaction partners, implying a gain in function. Our study elucidates adaptation of few SARS-CoV2 viral strains under the genomic compositional constraints during the outbreak shaped by the natural selection.


Despite large-scale quality improvement initiatives, substantial proportions of patients with ST-elevation myocardial infarction (STEMI) transferred to percutaneous coronary intervention centers do not receive percutaneous coronary intervention within the recommended 120 minutes. We sought to examine the contributory role of emergency medical services (EMS) activation relative to percutaneous coronary intervention center activation in the timeliness of care for patients transferred with STEMI.We conducted a retrospective analysis of interfacility transfers from emergency departments (ED) to a single percutaneous coronary intervention center between 2011–2014. We included emergency department (ED) patients transferred to the percutaneous coronary intervention center and excluded scene transfers and those given fibrinolytics. We calculated descriptive statistics and used multivariable linear regression to model the association of variables with ED time intervals  adjusting for patient age, gender, mode of arrival, weekday hour presentation, facility transfers in the past year, and transferring facility distance.We identified 159 patients who met inclusion criteria. Subjects were a mean of 59 years old (standard deviation 13), 22% female, and 93% White; 59% arrived by private vehicle, and 24% presented after weekday hours. EDs transferred a median of 9 STEMIs  in the past year and a median of 65 miles  from the percutaneous coronary intervention center. Median ED length of stay was 65 minutes . Among component intervals, arrival to ECG was 6%, ECG-to-EMS activation 32%, and ECG-to-STEMI alert was 49% of overall ED length of stay. Only 18% of transfers had EMS activation earlier than STEMI alert. ECG-to-EMS activation was shorter in EDs achieving length of stay ≤60 minutes compared to those >60 minutes . Multivariable modeling showed that after-hours presentation was associated with longer ECG-to-EMS activation . Female gender , prior facility transfers , and initial ambulance presentation  were associated with shorter ECG-to-EMS activation.In STEMI transfers, faster EMS activation was more likely to achieve a shorter ED length of stay than a rapid, percutaneous coronary intervention center STEMI alert. Large-scale quality improvement efforts such as the American Heart Association’s Mission Lifeline that were designed to regionalize STEMI have improved the timeliness of reperfusion, but major gaps, particularly in interfacility transfers, remain. While the transferring EDs are recognized as the primary source of delay during interfacility STEMI transfers, the contributions to delays at transferring EDs remain poorly understood. There is evidence that early initiation of emergency medical services (EMS) prior to activation of the cardiac catheterization laboratory at the percutaneous coronary intervention center may reduce the time spent at transferring emergency departments (ED).Vanderbilt University Medical Center (VUMC) is a quaternary care center for cardiovascular services in Middle Tennessee that provides 24/7 primary percutaneous coronary intervention capabilities and medical and surgical management of cardiovascular conditions. VUMC has a catchment area over 65,000 square miles and receives interfacility transfers from dozens of referring EDs in the region. Primary percutaneous coronary intervention activation at VUMC activates the cardiac catheterization team and prepares the laboratory for intervention. After hours, staff must be available onsite within 30 minutes. For the transferring ED, EMS must be activated separately and no formal policy exists regarding the order of such decisions.In this study we sought to examine the contributory role of each activity to overall STEMI transfer timeliness. We included patients with suspected STEMI who experienced interfacility transfer from an outside ED to VUMC between January 1, 2011–December 31, 2014. We excluded the following patients: 1) those who received fibrinolytics, which are recommended when the patient presents to a non-percutaneous coronary intervention facility and the anticipated delay to primary percutaneous coronary intervention is ≥120 minutes We developed a data dictionary and performed dual abstractor data collection using REDCap, a secure, browser-based, metadata-driven electronic data capture tool.What do we already know about this issue?Inter-facility transfer of patients with ST-elevation myocardial infarction (STEMI) are often prolonged due to coordination with emergency medical service (EMS) agencies and percutaneous coronary intervention (PCI) centers and impact patient outcomes.What was the research question?What is the contributory role of EMS versus PCI center activation in the timeliness of care for patients transferred with STEMI?What was the major finding of the study?Time spent at transferring EDs for patients with STEMI is more dependent on EMS activation than activation of the PCI center.How does this improve population health?Encouraging early EMS activation and incorporating this activity into formal policies may improve transfer timeliness for patients with STEMI.We calculated time intervals as the difference between two ED operational timestamps (identified above). When referencing ECGs, we used the ECG diagnostic of STEMI triggering the transfer. As some diagnostic ECGs may be performed by EMS prior to ED arrival, we set the arrival to ECG equal to zero as these visits had access to the diagnostic ECG upon arrival. Since we were using patients with suspected STEMI, and not all patients may have had stent placement, we used the timestamp for initiation of the percutaneous coronary intervention procedure, which was required for inclusion.pm and on weekends), number of facility transfers to VUMC for suspected STEMI in the past year at the time of transfer, and transferring ED facility distance. Analyses were conducted using R 3.5.1 .We calculated descriptive statistics for patient and facility characteristics, time intervals, and proportion of time intervals of the overall ED length of stay for the overall population, by EMS activation status (before vs after percutaneous coronary intervention activation) and by ED length of stay (≤60 minutes vs >60 minutes). We selected 60 minutes as the cutoff as this was the duration used internally for quality improvement purposes. Group comparisons were conducted using Wilcoxon rank-sum tests for continuous variables and chi-square tests for categorical variables. We used generalized linear models with log link function to quantify relative model ED time intervals of interest (ECG-to-EMS activation) adjusting for patient and facility characteristics, which included patient age, gender, ED mode of arrival , ED presentation after hours (>5 P = 0.047). However, ED arrival-to-percutaneous coronary intervention start was not different when EMS was activated first . Among transfers with an ED length of stay ≤60 minutes, 75% (N = 66) had an ECG-to-EMS activation ≤20 minutes. However, only 50% (N = 33) of such transfers with an ED length of stay ≤60 had an ECG-to-percutaneous coronary intervention activation that was ≤20 minutes. From an initial group of 439 subjects, we identified 159 patients who met inclusion criteria . SubjectP<0.001). Similarly, female gender  and increased interfacility transfers in the past year  were associated with shorter ECG-to-EMS activation  activating EMS earlier is more likely to reduce the amount of time spent at the transferring ED than percutaneous coronary intervention center activation; and 2) higher transfer volume in the past year, female gender, and after-hours presentations were associated with improved timeliness of EMS activation. These findings support policies that prioritize rapid EMS activation at the transferring ED. Further, these findings suggest that the increased interfacility familiarity that accompanies higher transfer volume may be a modifiable target for intervention to reduce STEMI transfer delays. Through reduction of transfer delays we seek to improve the timeliness of reperfusion as this is essential to optimizing patient outcomes.Our findings have practical implications for emergency clinicians who must transfer a patient with a suspected STEMI. This research provides evidence to support the clinician’s decision to activate EMS  prior to calling the percutaneous coronary intervention center. We found that EMS activation can be an important rate-limiting step in the timely transfer of patients with suspected STEMI. Three quarters of all transfers that had an ED length of stay less than 60 minutes had EMS activated in 20 minutes or less. This finding provides additional evidence supporting the use of early EMS activation in clinical practice.4The activation of EMS likely plays such an important role in the transfer process because the patient cannot leave until EMS arrives to physically transport the patient. On the other hand, while percutaneous coronary intervention center activation is important and necessary, the timing of this process appears to be less consequential. Although no formal policy exists at this study setting regarding the activation of EMS, in other settings, some EMS agencies still require an accepting physician name prior to transportation. Auto-acceptance protocols may work by simplifying the transfer process and improving the relationship between organizations by enhancing the likelihood that potential transfers will be accepted by the percutaneous coronary intervention center. As seen in this study, activating EMS prior to the percutaneous coronary intervention center activation was common practice despite no formal policy existing. In settings in which no formal policy exists regarding activating EMS prior to contacting the percutaneous coronary intervention center, incorporating such guidance into transfer center policies and protocols could be a strategy to enhance uptake of early EMS activation.We also identified that higher transfer volumes in the past year may also have reduced the time to activate EMS. More transfers may indirectly enhance the working relationship between facilities through organizational learning and improved timeliness. Research on interorganizational relationships also suggests that system membership and frequency of transfers may be related with timeliness of care.14Some limitations of this work should be considered. First, we conducted a retrospective analysis of patients with suspected STEMI transferred for primary percutaneous coronary intervention. Not all patients were ultimately diagnosed with STEMI, but the transferring ED and receiving percutaneous coronary intervention center operated as if it were a STEMI. This may, in part, account for our finding of improved timeliness in EMS activation for female patients who typically are less likely to receive percutaneous coronary intervention due to atypical presentations. Further, lack of severity at presentation may confound this finding. To enhance the quality of the retrospective data collection, we used dual abstractor review; however, transferring records might not have been available or potentially conflicting because organizational documentation and charting requirements might have been different . For exaTime spent at transferring EDs for patients with ST-elevation myocardial infarction is more dependent on activating emergency medical services rather than activation of the percutaneous coronary intervention center. Emphasizing this process and formally incorporating it into operational policies may improve transfer timeliness and subsequently reperfusion times.