Together with metabolites, proteins, and lipid components, the EV cargo consists of DNA and RNA nucleotide sequence species, which are part of the intracellular communication network regulating specific cellular processes and provoking distinct target cell responses. The extracellular vesicle (EV) nucleotide sequence cargo molecules are often investigated in association with a particular pathology and may provide an insight into the physiological and pathological processes in hard-to-access organs and tissues. The diversity and biological function of EV nucleotide sequences are distinct regarding EV subgroups and differ in tissue- and cell-released EVs. EV DNA is present mainly in apoptotic bodies, while there are different species of EV RNAs in all subgroups of EVs. A limited sample volume of unique human liquid biopsy provides a small amount of EVs with limited isolated DNA and RNA, which can be a challenging factor for EV nucleotide sequence analysis, while the additional difficulty is technical variability of molecular nucleotide detection. Every EV study is challenged with its first step of the EV isolation procedure, which determines the EV’s purity, yield, and diameter range and has an impact on the EV’s downstream analysis with a significant impact on the final result. The gold standard EV isolation procedure with ultracentrifugation provides a low output and not highly pure isolated EVs, while modern techniques increase EV’s yield and purity. Different EV DNA and RNA detection techniques include the PCR procedure for nucleotide sequence replication of the molecules of interest, which can undergo a small-input EV DNA or RNA material. The nucleotide sequence detection approaches with their advantages and disadvantages should be considered to appropriately address the study problem and to extract specific EV nucleotide sequence information with the detection using qPCR or next-generation sequencing. Advanced next-generation sequencing techniques allow the detection of total EV genomic or transcriptomic data even at the single-molecule resolution and thus, offering a sensitive and accurate EV DNA or RNA biomarker detection. Additionally, with the processes where the EV genomic or transcriptomic data profiles are compared to identify characteristic EV differences in specific conditions, novel biomarkers could be discovered. Therefore, a suitable differential expression analysis is crucial to define the EV DNA or RNA differences between conditions under investigation. Further bioinformatics analysis can predict molecular cell targets and identify targeted and affected cellular pathways. The prediction target tools with functional studies are essential to help specify the role of the investigated EV-targeted nucleotide sequences in health and disease and support further development of EV-related therapeutics. This review will discuss the biological diversity of human liquid biopsy–obtained EV nucleotide sequences DNA and RNA species reported as potential biomarkers in health and disease and methodological principles of their detection, from human liquid biopsy EV isolation, EV nucleotide sequence extraction, techniques for their detection, and their cell target prediction. With the development of deoxyribonucleotides and DNA molecules, more stable genomic information was generated and gene units for independent generation biological molecules of self-sufficient units were formed  in humans and other mammals include exosomes, microvesicles, and apoptotic bodies. All classes of EVs and their heterogeneous subsets differentiate in their biogenesis, diameter, and cargo, but all have a common feature of carrying and transporting parental cell molecules to target cells. Together with metabolites, proteins, and lipid components, the EV cargo consists of DNA and RNA molecules involved in the intracellular communication network regulating specific cellular processes and inducing distinct target cell responses . Human EIn the next chapters, we will briefly sum up the characteristics of EV nucleotide sequence species with their role and basic mechanisms in human cells and organisms. In the continuation, we describe the methods and procedures of EV nucleotide sequence biomarker discovery with methodological advantages and disadvantages and describe the prediction of EV nucleotide sequence cellular functions and their potential as therapeutics.The EV cargo consists of nuclear and mitochondrial DNA, while the EV transcriptome is more diverse and includes different coding and non-coding RNA species . The EV TP53, KRAS, and PTEN genes frequently altered in cancer and metastasis  were reported. Even though the mRNAs are not highly represented in EVs due to their size, EV mRNAs were reported in several studies which suggest the intracellular horizontal cell-coding transcriptome transfer . EV lncRhttp://www.mirbase.org/; Release 22.1) (The EV research is mainly directed into EV miRNA (microRNA) due to the well-characterized miRNA regulatory function in the cellular transcription and regulation of the mRNA expression to coding proteins. However, miRNA function is even more diverse. miRNAs are 16–24 nucleotides long sncRNA, coded by the miRNA genes which are in the human genome often located in miRNA gene clusters. 2,654 miRNA human sequences and 1,917 miRNA precursors are identified so far and their sequences are listed in miRBase (se 22.1) . It is ese 22.1) . The miRse 22.1) . The RISse 22.1) . miR-127se 22.1) , while sse 22.1) . Urinaryse 22.1) , while use 22.1) . The altse 22.1) .Piwi-interacting RNA (piRNA) regulation molecules are produced by similar synthesis, and have similar silencing mRNA translation and protein expression mechanisms as miRNA. This species of 26–31 nucleotides long sncRNA is highly expressed in germline cells and embryos, where pi-RNAs are involved in silencing of transposable elements, the epigenetic regulation, while in adult humans, piRNAs are essential in the process of spermatogenesis. EV piRNAs were investigated in seminal plasma EVs, where piRNAs are associated with spermatogenic ability , while mAnother sncRNAs reported in EVs are yRNAs, which are regulators of the DNA replication  and are EV transcriptome also includes tRNAs and tRNA fragments (tRFs), reported as highly represented in T-cell–released EVs . Even thSimilar to miRNA, other sncRNAs can be loaded into EVs together with RNA binding with Ago proteins or Y-box binding protein 1 , which avia miRNA-like silencing mechanisms, with binding on the mRNA transcription regulatory sites, other noncoding RNAs, or intracellular receptor mechanisms. If the EV RNA is released in the cytosol of the targeted cell, the released RNA can activate RNA-sensing receptors. Besides cytosol RIG1 receptors  columns, where vesicles are separated on a column by the EV diameter . The SECTissue-specific EVs can be isolated based on specific surface proteins with immunoprecipitation, where antibodies attached on immunoprecipitation columns or magnetic beads specifically bind to EV membrane protein markers . Due to All the EV isolation methods, some to a larger degree than others, also isolate non-EV impurity complexes. The most common isolated EVs from blood plasma contain EV low-density lipoproteins (LDL), high-density lipoproteins (HDL), and chylomicrones whose concentration is significantly increased in postprandial blood samples . TherefoWhile there are different EV isolation procedures, there are also diverse DNA or RNA isolation techniques based on different chemistry adjusted to isolate particular molecules of interest. The EV cargo typically consists of small DNA and RNA fragments; consequently, the isolation must isolate preferentially small nucleotide sequence fragments. The amount of the isolated EV DNAs or RNAs is highly variable to the isolation procedure, and it is the limiting factor that determines the EV nucleotide sequence characterization. A reasonable conclusion is that the EV DNA or RNA isolation method must ensure minimum sample losses.In order to ensure high purity and high yield of the isolated EV RNA or DNA, it is recommended to use the standardized, commercially available reagent kits for the EV nucleotide sequence extraction. The most common RNA and DNA isolation methods include the lysis reaction with the nucleotide sequence extraction, following precipitation and purification on the silicate columns and final elution . In the The EV nucleotide sequence content is still being discussed, but studies are unified in terms of a low EV nucleotide sequence quantitative amount. It was estimated that only one per 100 EVs contains miRNA . HoweverSeveral different methods can specify EV DNA and RNA molecules. The classic approach detects EV DNA or RNA using quantitative-PCR (qPCR), where only molecules of interest are analyzed, while the technique of massively parallel sequencing allows characterization of the whole RNA or DNA EV profiles . Due to Using quantitative PCR (qPCR), also known as real-time PCR, only the selected known sequences can be detected. EV sequences of interest can be selected based on the predetermined human organ and tissue expression data or can be predicted by known regulated pathways that are involved in the physiological and pathological conditions . The conEV transcriptome can also be detected using a microarray, where RNA fragments are directly labeled by the fluorescent dye and hybridized on predesigned probes with antisense sequences attached on a solid-phase glass surface or chip-based media. After wash steps, the fluorescence signal detection with intensity on specific locations on a glass slide is collected with a dedicated reader. The relative fluorescence of analyzed samples is translated to data with software algorithms, which recognize and quantify the studied particular sequences on the array surface . With miThe complete EV transcriptome and EV DNA profiles can be assessed only by NGS . Second-To simplify the sample sequencing preparation, several library preparation kits are on the market to prepare DNA, RNA, and sncRNA libraries. As small transcriptomic RNA molecules are abundant in EVs, it is recommended that small RNA library preparation kits are used for the NGS EV transcriptome detection . The smaNGS sequencing also presents the possibility to detect epigenomic modifications, which can be tissue-specific and can be very challenging in case of EV DNA and RNA. While the DNA methylation pattern detection is supported by the commercial reagents, sample preparation requires a large amount of DNA and analysis-specialized bioinformatics pipelines . The DNAThe third NGS generation is primarily designed as long-read sequencing technology for detecting DNA or RNA with several kilobase long fragments. This technology is suitable for detecting intact transcripts and providing accurate genome assemblies with successful detection even repeat sequences throughout the genome . Two comSince the EV sample processing and NGS library preparation require a skilled wet lab practice, the NGS sequencing data analysis requires bioinformatics knowledge to extract sequence information and address the EV nucleotide sequence study problem. The first part of the bioinformatics analysis starts with raw data base-calling translation from sequencing signals to nucleotide sequences. The base-called sequencing files are stored in the compressed fastq files generated by the Illumina sequencing platform and fastq5 files in the case of Oxford Nanopore. The signal translation base-calling output are text files with sequencing run info, nucleotide sequences, and quality of reading nucleotides .The RNA and DNA sequencing data analyses require different processing pipelines in the processing bioinformatics tools. However, the basic NGS pipeline includes the same basics steps: pre-processing and input data quality control, alignment, variant calling, quality control metrics report, and comparison of the expressed RNA analysis by the studied condition . First, Like in all the steps in EV research, the NGS data analysis procedure should be reported with used bioinformatics tools and their release number. It is also required to report basic analysis parameters and raw data quality parameters, sequenced reads per sample, and raw data should be submitted to public repositories, such as the SRA server (Sequence read archive), where sequencing data are accessible publicly to other researchers. While the NGS sequencing is still costly for assessing large cohorts, it is advised to confirm the NGS findings of smaller tested groups on larger cohorts, using the qPCR method.in vitro diagnostics (in vitro diagnostic EV liquid biopsy platform was presented ExoDx™ Lung (ALK) by Exosome Diagnostics. The kit detects EML4-ALK fusion transcripts in patients with non–small cell lung cancer, while the same company has also introduced exosome-based prostate cancer test ExoDx™ CE-IVD kit, which can help the physician’s decision to defer or proceed with a biopsy  includes siRNA-targeting transthyretin, used for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, which was first-of-its kind targeted RNA-based therapy to treat a rare disease (Several procedures allow the production of EVs with specific nucleotide sequence loading for potential therapeutic application. The nucleotide sequence–loaded EVs can be produced ectamine . The celantation . While E disease . The tec disease , malariain vivo mice animal model, enabling activation of gene transcription (While the synthetic vesicles are in use for the delivery of mRNA in vaccines, the vesicles can also be loaded with human RNA to substitute cellular-damaged mRNA sequences due to genomic mutations in coding genes. Futhermore, Lainšček et al. showed EV delivery of the CRISPR/Cas system with the transcriptional regulator in the cription . In addiFrom the first EV reports, where EVs were assumed as cell-released garbage, the EVs research revealed the complexity of EVs role in intracellular signaling and cell communication with their potential as biomarkers of physiological states and pathogenesis. In humans, most studies are focused on intracellular communication, even though EVs are also involved in the interkingdom vesicle communication between human cells and microorganisms or parasites. Gut bacterial microbiota EVs in the human organism can modulate metabolism, endocrine response, and consequently regulate the entire human organism with pro-inflammatory or anti-inflammatory response . Similarin silico differentially expressed EV-nucleotide sequences’ role in the organism, the excat biological function of these differentially expressed nucleotide sequences is often not verified with in vitro or in vivo experiments. In the future, more studies will try to characterize the EV nucleotide sequence biological effect. By collecting data from standardized compatible experimental setups, it would be possible to build deep learning algorithms and artificial intelligence in order to predict pathological differences. EVs automated processing and EV deep sequencing will make possible detection of pathological abnormalities even at early stages of the disease before the first clinical symptoms appear. With these conditions, the EVs would become reliable diagnostic markers and supportive diagnostic tools in clinical practice, driving the development of novel therapeutic approaches. The exciting field of EV nucleotide sequences research has a bright future. Advanced NGS methods with single molecule resolution will give RNA and DNA nucleotide sequences a whole new perspective and additional dimension by identification of nucleotide sequence modifications. Nowadays, the EV nucleotide sequences are commonly investigated only by their primary nucleotide sequence. While DNA nucleotide modifications can lead to gene silencing and mutation development, RNA modifications can have an essential role in cellular function regulation and immunomodulation and could be associated with wide plethora of diseases. Just as the presence of EV DNA modifications is not well understood, the same goes with the EV RNA modifications, although intracellular mechanisms of pathogen detection imply their potential function. The knowledge of the role of EV nucleotide sequence modifications will gain importance in the upcoming years and will probably reveal an additional regulatory layer of multiple biological functions and disease pathologies.More and more EV studies implement NGS in their study strategy and consequently, precise sample metadata annotation is imperative for reliable interpretation of EV-expressed nucleotide sequences. While most studies report The technology and methods in genetics are well under development, and the innovations in the methodological procedures will support low EV nucleotide sequences material input. Since the cellular nucleotide characterization techniques aim to define the expression profiles on a single cell level, similar characterization would be reasonably appreciated in EVs. Single vesicle nucleotide sequences with a simultaneous surface protein markers approach would give a whole new perspective on the EV’s complexity and diversity. Compared to present studies, single vesicle information would directly link the EV nucleotide sequences and proteomic profile to the EV-released tissues. Such an approach will allow the generation of EV multidimensional multi-omics data and facilitate a new specific biomarker discovery. Determining the roles of molecules inside vesicles and those on their surface represents another challenge for future characterization. The EV research is focused on the characterization of EV lumen nucleotide sequences, even though EVs can contain parental cell origin RNA and DNA attached on the EVs’ surface lipids or anchored by glycosylated anchors .ex vivo or even in vivo treatment for inborn genetic disorders and could substitute ex vivo cell treatment for somatic mutations with viral vectors. While the lipid nanoparticles are preferentially accumulated in the lymph nodes, spleen and lungs, the future of EV therapeutics is in specific EV target tissue delivery. This could thus be achieved by EV-specific target membrane surface proteins (Because of the regulatory role of EVs, their therapeutic possibilities are being discussed and some preclinical and clinical trials already show promising results. Autologous platelet-produced therapeutic EVs significantly improve chronic postoperative healing and regenerative medicine , while mproteins , lipids,EVs and their nucleotide sequences, with their diversity, present an exciting research field that is still practically undiscovered and with enormous innovation potential represent one of the most promising fields of biomedical research.


The expanding armamentarium of wearable activity monitors (WAMs) offers new opportunities to supplement physician-assessed performance status (PS) with real-life patient activity data. These data could guide clinical decision making or serve as a measure of treatment outcome. However, information on the association between physical activity (PA) and sedentary behavior (SB) monitored with wearables  and PS in patients with cancer is needed. Therefore, we conducted a systematic review to examine the association between WAM metrics and PS in patients with cancer.We searched MEDLINE and Embase for studies that assessed the association between WAM metrics and performance status among adults with cancer. We extracted information on study design and population, WAM type and different activity metrics, outcome definitions, and results. Included studies were subjected to risk of bias assessment and subsequent best evidence synthesis.n = 8), SB (n = 5), mean activity counts (n = 4), dichotomous circadian rest-activity index (n = 3), and time spent in moderate-to-vigorous PA (MVPA) (n = 3). Much heterogeneity was observed regarding study population, WAM used, and reporting of results. We found moderate evidence for a positive weak-to-moderate association between WAM-assessed PA and PS and a weak-to-moderate negative association between WAM-assessed SB metrics and PS.Fourteen studies were included in this review. All studies reported on different combinations of WAM metrics including: daily steps (Weak-to-moderate associations between WAM metrics and PS suggest that WAM data and physician-assessed PS cannot be used interchangeably. Instead, WAM data could serve as a dynamic and objective supplement measurement of patients’ physical performance.The online version contains supplementary material available at 10.1007/s00520-021-06234-5. Patients’ performance status is a significant prognostic and predictive factor for clinically relevant outcomes, such as progression-free and overall survival of patients with cancer –3. It thThe expanding armamentarium of wearable activity monitors  offers new opportunities to supplement physician-assessed performance status with objective assessments of physical activity and sedentary behavior, which are passively gathered in a non-obtrusive manner. It is even hypothesized that wearable activity monitor metrics might prove superior to clinician-rated performance status or patient-reported data in terms of accurately discriminating between the heterogeneous spectrum of cancer patients , 14. TheA systematic review of available literature was conducted in agreement with the guideline for preferred reporting items for systematic reviews and meta-analyses (PRISMA statement) . This reMEDLINE® and Embase databases were searched from inception until April 2020 to identify all relevant published articles. An experienced clinical librarian from the Amsterdam UMC was consulted for the development of the search strategy. Relevant keywords included terms related to wearable activity monitor metrics AND cancer population AND wearable activity monitors . The complete search strategies are presented in Supplementary Table Studies were included if they (1) were conducted among adults (≥ 18 years) with cancer, (2) objectively measured physical activity or sedentary behavior using wearable activity monitors in the outpatient setting, (3) measured physician-assessed performance status, (4) quantitatively assessed an association between wearable activity monitor metrics and clinician-assessed performance status, and (5) had a full text available in English.Many different activity-related wearable activity monitor metrics are being used in research and reported wearable activity monitor metrics often depend on the used device. Four main categories of wearable activity monitor metrics relevant for this review can be identified: (1) accelerometer-related activity count-based metrics that capture the duration and intensity of accelerations in counts per minute and/or hours. Moreover, these intensities can be used to determine absolute or relative time spent in sedentary behavior, light physical activity (LPA), or moderate-to-vigorous physical activity (MVPA) based on predefined cut-points. (2) Posture-based measures that define hours of percentage of time per day spent sitting/lying , standing, or stepping. (3) Steps-based measurement that estimate the number of steps per day using an algorithm that determines whether accelerometer measurements meet the threshold to be counted as a step. (4) Circadian rhythm rest-activity actigraphy parameters like, the dichotomous in-bed vs out-of-bed index (I < O) and mean activity level (MeanAct).Titles and abstracts of articles identified by the electronic database searches were extracted and duplicates were removed. Two reviewers (MK and EP or BB) independently screened the records of the initial search to select potentially relevant articles that were subsequently subjected to full-text screening for eligibility. Any discrepancies between reviewers were discussed in person. If no agreement was reached, discrepancies were referred to a third reviewer (MvO) before a final decision was made on inclusion.Data on study design and population, physical activity/sedentary behavior measurement characteristics and protocols, wearable activity monitors, and outcome measures were extracted using a standardized form including the following items: first author, year of publication, country, study design, number of included patients, type of cancer and disease stage, current treatment, comorbidities, performance status scale used, measure of physical activity or sedentary behavior (including definitions and cutoff points), devices used for physical activity/sedentary behavior measurements, wear location of devices, statistical methods and analyses, and results on association between wearable activity monitor metrics and performance status. If point estimates  were only depicted in figures, authors were first contacted and asked to provide these point estimates. If we did not receive data from the authors, we used open source software WebPlotDigitizer (version 4.3) to estimate the point estimates and corresponding measures for variability  from the figures –20. The The risk of bias of included studies was scored independently by two reviewers (MK and EP) using a risk of bias assessment tool based on the guideline for assessing quality in prognostic studies . These gA 3-level scoring system for best evidence synthesis based on the number, methodological quality, and consistency of outcomes of the different studies was applied to synthesize the methodological quality of the included studies , 27, 28:. After initial screening, 373 articles were retrieved in full-text and checked for eligibility. Cross-referencing provided one additional study [. Ultimately, 14 studies met the eligibility criteria and where included for further analysis. Table The combined literature search yielded 1511 unique records. The result of the systematic literature search and subsequent selection of studies is depicted in Fig. 1al study  that coun < 100). None of the studies adequately described methods used for dealing with missing physical activity data, and the majority of studies (79%) used a combination of device and wear-time protocol that has not been adequately validated in the studied population.Results of the risk of bias assessment are depicted in Table The characteristics of the used wearable activity monitors and physical activity/sedentary behavior metrics are summarized in Table Table In total, we found 14 studies that could be included in the evidence synthesis. Results of evidence synthesis for the association between wearable activity monitor metrics and performance status are compiled in Table In this study, we reviewed the available evidence on the association between wearable activity monitor metrics and physician-assessed performance status. Evidence synthesis showed moderate evidence for weak-to-moderate positive associations between performance status and various wearable activity monitor metrics and a moderate negative association between performance status and sedentary behavior.Different possible explanations can be provided for the absence of strong associations. First, these weak-to-moderate associations may suggest that wearable activity monitors and performance status scales assess different constructs of physical performance and cannot simply be interchanged. Wearable activity monitors objectively measure physical activity (levels) and can therefore be regarded as performance-based measurements that are independent of judgment. Performance status scales, on the other hand, are evaluation-based measurements that involve judgment using idiosyncratic criteria , 6. AnotMore detailed objectively and passively gathered activity data from wearable activity monitors might be of added value in clinical practice. Wearable activity monitor-assessed physical activity/sedentary behavior might serve as a dynamic and objective supplement measurement of patients’ performance status as assessed by the physician and, as such, might prove to be of added value in clinical decision making and evaluation of treatment options in oncology. This hypothesis is substantiated by observations that more daily steps are associated with lower risk of hospitalization during cancer treatments , 43, lonMost currently available wearable activity monitors  are multisensory devices that have a built-in 3D-accelerometer as well as other sensors that measure, for example, heart rate. In the era of advancing artificial intelligence and machine learning, it is very conceivable that data input from combinations of wearable activity monitor sensors and data patterns over time might prove to be superior in assessing performance status and predicting outcomes for patients with cancer than physician-assessed performance status.Recently, various pilot studies have demonstrated the feasibility of using wearable activity monitors in the context of an ambulatory monitoring platform that longitudinally assesses treatment-related adverse events, unplanned healthcare encounters, and survival in patients with cancer , 46, 47.An important finding of our systematic review is the high heterogeneity between included studies regarding study population, devices used, wear-time protocols, definitions and cut-points used for different physical activity metrics, and reporting of outcomes, thereby hindering adequate comparison of results on the association between physical activity metrics and performance status and complicating best evidence synthesis. A second limitation of the studies included in this review is the high risk of bias scores. Major factors contributing to the high risk of bias scores were unvalidated methods regarding physical activity measurements, low sample sizes, and the lack of multivariable analyses to adjust for relevant confounders. It should be noted that the majority of these studies investigated the association between wearable activity monitor metrics and performance status as secondary or exploratory analysis, which may have contributed to the high risk of bias scores. Consequently, it is currently also unclear whether the association between wearable activity monitor metrics and performance status varies by cancer type or stage. With regard to the physical activity measurements, none of the studies adequately reported on the handling of missing physical activity data. Different studies have emphasized the need for missing accelerometer data imputation and suggested statistical methods of handling missing data , 49. MorStrengths of this systematic review include the in-depth risk of bias assessment that was adjusted specifically for studies using a wearable activity monitor for physical activity and sedentary behavior measurements and the subsequent best evidence synthesis. However, most of the included studies were not designed to investigate the association between wearable activity monitor metrics and performance status, complicating risk of bias assessment and evidence synthesis. More than half of the included studies were designed to investigate physical activity levels in specific cancer populations, study the feasibility of wearable activity monitors, or explore associations between other wearable activity monitor metrics, like circadian rest-activity rhythm parameters, and various outcomes. Therefore, the association between wearable activity monitor metrics and performance status was often analyzed in a secondary or exploratory analysis resulting in suboptimal presenting of results. Moreover, results may be prone to reporting bias as non-significant associations are less likely to be reported, resulting in an overestimation of the associations between wearable activity monitor metrics and performance status.In conclusion, we found moderate evidence for a positive weak-to-moderate association between various physical activity metrics and performance status and for an inverse moderate association between sedentary behavior and performance status. The strength of the associations should be interpreted with caution given the aforementioned limitations of the available evidence. Nevertheless, our results suggest that objectively measured physical activity may serve as a dynamic and objective supplement measurement of a patient’s functional performance status and may be of added value in clinical decision making and evaluation of treatment options in oncology. Next steps include to study the association between wearable activity monitor metrics and clinical outcomes and directly compare the predictive value of objectively measured physical activity with performance status for relevant clinical outcomes. Finally, consensus is warranted on the methodology of objective physical activity measurement and efforts should be made to validate the different methods  in relevant patient populations.Supplementary file 1 (PDF 91 KB)Below is the link to the electronic supplementary material.


There is evidence to support the idea that bacteria and archaea form cohesive sequence clusters  at a particular multidimensional environmental space. However, the microbial population interacts with the niche, creating sub-niches that are colonized by variant sub-populations of bacteria. Such a diversification process is compensated by the genetic interactions of sub-niche bacterial variants, leading to an evolved population able to fully exploit the expanded niche. Optimization occurs when the full carrying capacity of the niche is reached, and such perfect niche-bacteria trade-off facilitates specialization and eventually genetic isolation and speciation. In summary, the origin of species and the origin of niches are entirely intertwined.In the article entitled “Insights on the Evolutionary Genomics of the Blautia Genus: Potential New Species and Genetic Content Among Lineages,” Maturana and Cárdenas used a pangenomic analysis combined with a whole-genome approach to reevaluate a previously known taxonomic group, which both discovered new species and reassigned others. Analysis of hundreds of publicly available Blautia strain genomes found 17 previously undescribed species and helped to reconstruct important gene gain/loss events during the diversification of the Blautia genus, shedding light about how this genus specialized to live in the gastrointestinal tract.The article entitled “Comparative Genomic Insights into the Taxonomic Classification, Diversity, and Secondary Metabolic Potentials of Kitasatospora, a Genus Closely Related to Streptomyces,” Li et al. used comprehensive phylogenomics and comparative genomics analyses to examine genomic differences among lineages within the genus Kitasatospora. It revealed genomic differences between the closely related genera Streptomyces and Streptacidiphilus, supporting Kitasatospora as a separate genus within the family Streptomycetaceae. Comprehensive annotation also uncovered unexpected potentials of secondary metabolism in Kitasatospora, which carries more and longer secondary metabolite biosynthetic gene clusters than the well-known antibiotic producer Streptomyces.The article entitled “Phylogenetic Distribution of Polysaccharide-Degrading Enzymes in Marine Bacteria,” Sun et al. used large-scale genomics to annotate polysaccharide-degrading enzymes in marine bacterial genomes based on a manually curated enzyme sequence database. The authors found extended phylogenetic distributions for most enzymes analyzed. Interestingly, there was substantial intra-clade diversity in the coding potential of polysaccharide-degrading enzymes, indicating diversification in the polysaccharide-utilization abilities and thus niche differentiation. The study suggested that the polysaccharide-degrading enzymes could be used as molecular markers for marine bacterial niche adaptation and speciation.The article entitled “Host- and Species-Dependent Quasispecies Divergence of Severe Acute Respiratory Syndrome Coronavirus-2 in Non-human Primate Models”, Hwang et al. examined newly emerging SARS-CoV-2 variants that have been reported to cause higher morbidity and mortality than previous viruses despite enforcement of strict biosecurity and vaccination policies. Based on single nucleotide polymorphism analysis, the high intra-host, inter-host and inter-species genomic variability of SARS-CoV-2 following a short replication period were verified in Cynomolgus monkeys and Rhesus macaques infected with identical SARS-CoV-2. These results are valuable not only for understanding SARS-CoV-2 variant evolution in hosts, but also for development of new diagnostics, vaccines, and therapeutics targeting SARS-CoV-2.The article entitled “Comparative genomics of novel Agrobacterium G3 strains isolated from the International Space Station and description of Agrobacterium tomkonis sp. nov.,” Singh et al. used a polyphasic approach to characterize the phenotypic and genotypic synapomorphies of a novel Agrobacterium tomkonis, showing it is a bacterial species, well-separated from previously named Agrobacterium species. A. tomkonis are diverse and have been isolated from hospital, plant rhizosphere soil, and the International Space Station surfaces. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved in the ecological specificity of A. tomkonis, as suggested by their varied source of isolation. The ability to colonize nutrient-poor substrates, unusual for most agrobacteria, resonate with the finding that A. tomkonis genomes specifically carry genes involved in mediating attachment to surfaces, including production of putative adhesins and biofilm production. This suggests a particular ability of A. tomkonis to colonize different habitats that are even poorer in nutrients and harsher than soil, thus possibly escaping competition with other agrobacteria that are better at growing in richer environments, such as plant rhizospheres.The article entitled “Using modern sequencing technologies and bioinformatic techniques, researchers observed patterns of microbial diversity across multiple taxa and in numerous distinct systems. Observations were made of microbial populations evolving to unique ecological niches, regardless of taxa or environment. While horizontal gene transfer can allow for niche adaptation and act as cohesive force among populations, the observed populations appeared to have diversified though selection to unique, and sometimes subtle, ecological niches. Continued research combined with improving technology will allow researchers to determine if a common species definition can ultimately be applied across all bacterial and archaeal lineages.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ClinicalTrials.gov, were searched for publications in the last 20 years. We included 14 studies, seven randomized control trials, and seven post hoc analyses in this paper. Atrasentan decreased albuminuria, reduced blood pressure, and improved lipid profiles with more manageable fluid overload-related adverse events than avosentan and bosentan. Overall, endothelin receptor antagonists, in combination with renin-angiotensin-aldosterone system inhibitors, effectively reduce albuminuria and prevent the progression of diabetic kidney disease. However, more extensive clinical trials still need to be conducted to confirm these relationships and to learn more about the specific factors affecting their efficacy in individual patients.Diabetic nephropathy is becoming a more predominant cause of end-stage renal disease, as the prevalence of diabetes mellitus worldwide is on the rise. In this systematic review, we aimed to define the role of endothelin receptor antagonists, in the prevention and treatment of diabetic nephropathy, in addition to determining their safety. For this review, PubMed, Google Scholar, and Cochrane Library databases, in addition to  According to the World Health Organization (WHO), approximately 422 million people suffer from diabetes mellitus worldwide, regardless of their economic status . With thFor over two decades, the standard treatment of diabetic nephropathy has focused on strict glycemic control and blood pressure control with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) . AlthougEndothelin (ET) receptor antagonists are a promising group of drugs for reducing albuminuria in patients with diabetic kidney disease. In fact, there are no published studies of a medication added to RAAS antagonists that reduces albuminuria as effectively as ET receptor blockers . There aThere has been plenty of research identifying the role of ET receptor antagonists in the prevention of diabetic nephropathy-associated albuminuria in experimental studies. More recently, several randomized clinical trials (RCTs) have been conducted to ascertain the role of these drugs in humans. ETA selective receptor antagonists prevent inflammatory and cytoskeletal changes in podocytes induced by ET-1. ETB receptor blockade is associated with higher adverse effects such as sodium and water retention and decreased nitric oxide production. Therefore, more clinical trials have focused on ETA selective blockade with atrasentan (ETA: ETB blockade ~1200:1) or avosentan (ETA: ETB blockade ~50-300:1), rather than non-selective blockade with bosentan (ETA: ETB blockade ~20:1) . HoweverMethodsThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement  was usedSearch Strategy:ClinicalTrials.gov was accessed to retrieve relevant clinical trials. A comprehensive data search was performed using the databases PubMed, Google Scholar, and Cochrane Library. The databases were searched for papers published in the last two decades, from January 1, 2001, to June 20, 2021. Medical Subject Heading (MeSH) terms and the following keywords were searched in various combinations: "Diabetes mellitus," "Endothelin receptor antagonists," Bosentan, Avosentan, Atrasentan, "Diabetic nephropathy," and proteinuria. In addition, Study Screening and Selection:The titles and abstracts of the search results were screened for relevancy of information after the removal of duplicate articles. The remaining publications were then individually screened by two reviewers, using the following inclusion criteria:(1) Studies published between 2001-2021(2) Studies published in the English language(3) Human studies(4) Randomized control trials comparing endothelin receptor antagonists with placebo(5) Studies performed in diagnosed diabetic patients (aged ≥18 years), with evidence of proteinuria and decline in glomerular filtration rateStudies in languages other than English, animal research studies, studies involving non-diabetic patients, and studies that were not randomized control trials were excluded. Data Extraction and Quality Assessment:Two researchers independently evaluated the whole text of the remaining publications to extract data. The author names, year of publication, study design, sample size, interventional drugs, and study outcomes were noted. The same reviewers then assessed the quality of the final eligible studies using the Cochrane risk of a bias tool for RCTs . SelectiResultsLiterature Search:ClinicalTrials.gov. After removing duplicate studies and screening the titles and abstracts, 54 articles remained. Eventually, 14 studies were included in this systematic review, of which seven are post hoc analyses. Figure A total of 824 articles were initially identified through our search of PubMed, Google Scholar, Cochrane Library, and Characteristics of the Included Studies:The following 14 studies were included in this review: Wenzel et al. , Mann etAll RCTs evaluated the efficacy and adverse effects of various doses of the interventional drug versus placebo. The participants of all studies were diagnosed as people with diabetes showing clinical evidence of albuminuria and were already on stable doses of ACE inhibitors or ARBs. However, in the study on bosentan, 19 out of 24 participants and 20 of 22 participants in the interventional and control groups, respectively, were taking RAAS inhibitors . The effRisk of Bias Assessment:Figures DiscussionBased on the available clinical data on the use of endothelin receptor antagonists, it is evident that these agents are a promising new hope for the prevention and treatment of diabetic-related kidney disease. The more ETA-selective receptor agents, atrasentan and avosentan, have shown more significant results in reducing albuminuria in high-risk diabetic individuals than bosentan, a dual ETA-ETB receptor antagonist. ETA receptor blockade leads to reduced glomerular vasodilation, altering the glomerular permeability for albumin and thus, lowers the tubular load of albumin . This, iHowever, the ET receptor antagonists are not without adverse effects. There is an indication of a higher risk of cardiovascular and edema-related events occurring with these drugs than placebo. The most commonly reported side effects are edema, hypervolemia, hypotension, anemia, dyspnea, hypoglycemia, and headache ,32. CardEdema was reported with all three drugs, but there was no significant difference when treatment groups were compared to placebo in a meta-analysis . These fAn important observation that was drawn from past clinical studies is that the dose-response associated with fluid retention is different from the dose for the albuminuria-lowering effect . One posBosentan:Although bosentan did not show significant results of decreasing UACR, Rafnsson et al.  found thDigital endothelial function was measured via pulse amplitude tonometry to determine the change from the baseline of the reactive hyperemia index (RHI). Flow-mediated vasodilation (FMD) of the brachial artery was also determined to assess macrovascular function. RHI was increased in the bosentan group, but the brachial artery FMD did not change, indicating that bosentan improves endothelial function in only small vessels and therefore plays a role in preventing the microvascular changes of diabetes mellitus. As nitroglycerine-induced digital hyperemia was not affected, it was concluded that bosentan did not play a role in endothelium-independent vasodilation .An interesting finding was that plasma ET-1 levels were mildly increased in the treatment group compared to placebo when they would be expected to decrease with an ET receptor antagonist. This phenomenon could be attributed to the fact that the ETB receptor is responsible for the clearance of ET-1. This theory was supported by Andress et al.'s  trial fiThe adverse effects of bosentan could not be fully appreciated in this small, short-term trial. Three patients withdrew from the trial due to adverse effects, and only one of them had edema. Since there have not been enough clinical trials studying bosentan, it is hard to determine its safety. However, its possible role in preventing microvascular changes should be further studied in more extensive trials. A significant drop in hemoglobin was found, as seen with avosentan and atrasentan.Avosentan:Wenzel et al.'s  trial waEven though Wenzel et al.  discoverIt is believed that avosentan becomes less selective for the ETA receptor at higher doses and leads to sodium and fluid retention effects of ETB receptor blockade, which also explains the slight increase in body weight. Whether the edema-related adverse effects were due to high dosages or to the participants having stage III/IV, CKD is still uncertain requires more research to ascertain the safety of avosentan .The role of avosentan in affecting GFR is unclear as the GFR declined in both avosentan treatment groups as well as the control group. However, there was a more considerable decrease in GFR reported in the 50mg group, which could be attributed to drug-induced fall in intra-glomerular pressure. Although ERSD occurred less frequently with avosentan, it could have been due to early termination of the trial and incomplete results. Overall, mild blood pressure reduction was noted, and lipid profiles were improved with avosentan administration ,20. HighAtrasentan:More clinical research is available on atrasentan, perhaps due to its higher selectivity for the ETA receptor and its decreased risk of adverse effects at moderate doses. Zhou et al.  found thAccording to Kohan et al. , GFR didWebb et al.  conducteHemoglobin was significantly reduced in the ET receptor antagonist group (atrasentan and avosentan), as compared to the control, and there was an increased risk of anemia for the treatment group ,24,29. TFactors Affecting ET Receptor Antagonist Response:Overall, Caucasian patients had greater albuminuria lowering effect than Black patients . The phaA post hoc analysis of the enrichment period of the SONAR trial reported that there was high variability in the albuminuria lowering effect and BNP altering of atrasentan between patients ,31. ThesIt was found that those with higher baseline albuminuria and lower baseline BP would better benefit from the albuminuria reducing the effect of atrasentan . On the Pena et al.  studied ET Receptor Antagonists in Combination With Other Drugs:Patients receiving maximum doses of RAAS inhibitors had a similar response to those receiving other doses, which might imply that patients do not need to be on maximum therapy after all . This caET Receptor Antagonists in the Future:Other factors may affect the efficacy of ET receptor antagonists that are yet to be explored. As Yuan et al.  pointed Schievink et al.  suggesteLimitations:Some of the trials included in this systematic review had few participants, which means that the results could largely depend on the larger-scale trials. The duration of treatment and follow-up periods also varied in trials and could have affected outcomes and adverse events reporting. The research on ET receptor antagonists is still limited as more studies have been conducted on atrasentan than avosentan or bosentan. Early termination of the ASCEND and SONAR trials leading to shortening of follow-up could have also provided incomplete results. Trials that excluded patients for whom the drug may not be beneficial may have been favorable in terms of preventing adverse effects; however, it is uncertain whether the approach to omit those patients was correct.Endothelin receptor antagonists have shown high efficacy in reducing albuminuria in patients with diabetic kidney disease. More specifically, there is greater evidence of atrasentan demonstrating enhanced effects on albuminuria reduction than avosentan and bosentan, with lower doses also causing minimal adverse events. In addition to this favorable effect on albuminuria, ET receptor antagonists also play a role in blood pressure control and improve lipid profiles. This systematic review helps in establishing a beneficial role of the ET receptor antagonists. However, to further prove the effectiveness of these drugs, more research is still required. This review highlights the inter-individual variations in drug effects based on various factors, such as demographic and lab values. These differentiating factors may be used to conduct additional studies using specifically designed methods to select the most appropriate participants and doses of the drugs. Planning in these regards is necessary in order to achieve better results while also preventing specific adverse effects.


The use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 has moved from bench to bedside in less than 10years, realising the vision of correcting disease through genome editing. The accuracy and safety of this approach relies on the precise control of DNA damage and repair processes to achieve the desired editing outcomes. Strategies for modulating pathway choice for repairing CRISPR-mediated DNA double-strand breaks (DSBs) have advanced the genome editing field. However, the promise of correcting genetic diseases with CRISPR-Cas9 based therapies is restrained by a lack of insight into controlling desired editing outcomes in cells of different tissue origin. Here, we review recent developments and urge for a greater understanding of tissue specific DNA repair processes of CRISPR-induced DNA breaks. We propose that integrated mapping of tissue specific DNA repair processes will fundamentally empower the implementation of precise and safe genome editing therapies for a larger variety of diseases. Genome stability is constantly challenged by endogenous and exogenous factors that threaten the integrity of DNA. If DNA damage is incorrectly repaired, this leads to mutations or wide-spread genome aberrations that impair cell function and survival. Intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), reactive metabolites, and replication stress synergise with exogenous genotoxic sources of damage, such as radiation, chemical exposure, viral, or bacterial infections to challenge genomic stability. In order to protect genome integrity, cells have evolved sophisticated mechanisms to detect, signal, and repair diverse DNA lesions, known as the DNA damage response.via topoisomerase inhibition. In contrast, formation of endogenous DSBs is an integral part of fundamental cellular processes, such as the generation of immune receptor diversity, meiosis, and ageing (DNA double-strand breaks (DSBs) are amongst the most toxic lesions cells can encounter, as both DNA ends become topologically separated. For this reason, DSBs are induced in cancer therapy, either through ionising radiation or by preventing their repair d ageing . Therefod ageing . A key aStreptococcus pyogenes Cas9 (SpCas9), generating a blunt end at a targeted genomic site. We direct readers to the following additional technical advances that have expanded the CRISPR-toolbox and fall outside the focus of this review: engineered Cas9 nucleases with higher fidelity  and transcription activator-like effector nucleases (TALENs) have been successfully used as genome editing tools in mammalian cells . Howeverfidelity  and broafidelity , DSB-indfidelity  and primfidelity , CRISPR-fidelity , and newfidelity .via end-resection, is the major factor dictating repair pathway choice. Although HDR faithfully repairs lesions, the end-joining pathways are preferentially upregulated through several mechanisms following DSB formation. This is because NHEJ is active throughout all phases of the cell cycle, predominating in G0 and G1  and Exonuclease 1  within the targeted genomic sequence. If these indels give rise to frameshift mutations, they result in loss-of-function alleles. This type of repair outcome has been largely attributed to the use of NHEJ, which directly ligates the two DNA ends following cleavage, leading to the generation of small indels <10bp; . More re 1 EXO1; .Albeit being generally considered as an alternative pathway, studies based on the pharmacological and genetic ablation of NHEJ have shown that MMEJ can fully compensate for the absence of NHEJ in the repair of Cas9-induced DSBs . DespiteIn contrast to the end-joining pathways, and within the context of genome editing, HDR depends on an exogenous repair template, allowing cells to integrate specific and precise alterations in their genome , thus maImportantly, transcriptional and genetic differences impact the efficiency of CRISPR-Cas9 editing and therefore the effectiveness of genome editing approaches. Screens performed in human cancer cell lines have shown that the Fanconi anaemia (FA) pathway diverts repair toward SSTR, playing an important role in HDR efficiency . The FanThe importance of DNA repair for genome editing applications is further illustrated by the different approaches that modulate DNA repair pathways to improve HDR efficiency. For example, since NHEJ is the default pathway in human cells, its inhibition has been exploited to favour HDR. This has been achieved through the use of small-molecules targeting LIG4 or DNA-PKcs , ubiquitThe DNA damage response is a highly interconnected signalling network, which is modulated by cell cycle stage, gene expression changes, chromatin states, differentiation status, and cell type .ex vivo edited cell populations  technologies have empowered researchers to look for off-target effects beyond commonly predicted sites, enabling high standards for quality control of ulations . Even inA recently developed approach termed Repair-Seq was used to systematically map DNA repair outcomes, and hence editing outcomes, after Cas9 and Cas12a mediated genomic editing across several loci . This reIn summary, recent insights into the complex interplay between DNA break configuration and DNA repair factors, highlighted how the landscape of genome editing outcomes remains underexplored. The studies discussed above made their observations in a few cellular models but found a surprising variety of lesions and repair outcomes generated. The level of complexity further increases when one takes cell type and tissue specific effects of DNA repair into consideration. It becomes apparent that the full control of CRISPR-mediated genome editing is only possible with full understanding of the intricacy of endonuclease generated lesion conformation in combination with DNA repair regulation in a tissue dependent context.BRCA1/2 mutations, which cause a defect in HDR, yet predispose primarily to breast and endometrial cancers. Similarly, defects in DNA single strand break repair (SSBR), predominantly affect neuronal cell types, while, for instance defects in crosslink repair (Fanconi anaemia pathway) precipitate bone marrow failure and neurological degeneration  and organoid-based approaches  gene. The function of this chloride/bicarbonate channel is to regulate the exchange of electrolytes and thus the hydration levels of secretory epithelia. Loss or reduction of function in this protein leads to cycles of mucus accumulation, inflammation, and infection in the lung, progressively destroying the airway epithelium  amyloidosis, and heritable blindness, CRISPR-therapies have become available to patients. We review recent achievements in clinical trials and consider the applicability of tissue specific DNA repair.ex vivo therapies to patients and demonstrated safety and feasibility of these treatments. Yet, these trials have also demonstrated that the mere reduction of off-target editing is not sufficient to achieve the desired outcome. One trial (NCT02793856) studying the therapeutic effect of knocking out the programmed cell death protein 1 (PD-1) in patient derived T-cells via NHEJ in refractory non-small-cell lung cancer, found a good ratio of 48.7 of on-target over off-target editing. Even so, 28.8% of all on-target edits did not match the predicted outcome . Targeting CD45-positive hematopoietic stem and progenitor cells, the moglobin . Precisemoglobin . Howevermoglobin . In futumoglobin .via delivering CRISPR-Cas9 along with a single-stranded DNA repair template into hepatocytes, using hydrodynamic tail vein injection. Once stimulated to proliferate, actively cycling hepatocytes can utilize HDR to make the edit of interest. Although only one in 250 liver cells were successfully edited, corrected cells have a selective advantage and begin to outgrow unedited cells and repopulate the liver, effectively ameliorating the disease. Therefore, considering tissue architecture along with DNA repair pathway choice, results in a therapy which is more effective than the initial editing efficiency.Integrated knowledge of tissue architecture and DNA repair outcomes can help designing better CRISPR therapies. A prime example of this is the fatal genetic disease hereditary tyrosinemia type I (HTI). HTI is caused by a G>A point mutation in the fumarylacetoacetate hydrolase (FAH) gene, which causes skipping of exon 8, leading to a dysfunctional protein and accumulation of the toxic metabolite fumarylacetoacetate in hepatocytes, ultimately leading to cirrhosis, acute liver failure, and increased risk of hepatocellular carcinoma . The livin vivo editing (Gene editing of hepatocytes has recently found application in a clinical trial using  editing . TTR amy editing .in vivo therapy targeting differentiated cells. It consists mostly of intermittently mitotic hepatocytes, which at once reduces the risk of pathogenic outgrowth, compared to consistently cycling cells, and simplifies the complexity of having to consider many cell types in the design of the editing strategy. Aside from the rarity of hereditary ATTR, pathogenic accumulation of wild type TTR fibers in the heart is also observed in patients and has been recognised as a cause for cardiomyopathy and eventual heart failure ( failure . Hence, in vivo CRISPR Cas9 editing. One trial is seeking to treat Leber congenital amaurosis (LCA; via generating an indel through NHEJ (in vivo CRISPR editing begins to move into the clinic, it is pertinent to keep in mind that LCA constitutes an ideal model disease for this approach. The post-mitotic nature of the targeted cells ensures a greater propensity for utilising NHEJ to repair the induced break and reduces the risk of selective pathogenic outgrowth of edited cells, when compared to actively cycling somatic stem cells. Furthermore, there is reduced risk of inflammation or adverse reactions to introduction of Cas9, due to the immunoprivileged status of the eye.Since specificity of editing outcomes and safety are still major technological hurdles, there are currently few ongoing clinical trials utilising sis LCA; . LCA mansis LCA; . The CRIugh NHEJ . While iThe examples above illustrate the potential and versatility of CRISPR-based therapies. The success of such approaches, however, relies on careful consideration about the biology of targeted cells and a deep understanding about the tissue specific mechanisms of DNA damage signalling and repair.The successful implementation of CRISPR-Cas9 technologies in a clinical setting relies on a deeper understanding of the DNA repair mechanisms and pathways responsible for genetic replacement outcomes, as well as the activity and accessibility of these pathways in specific cell types and tissues. Following the generation of a DSB, cell cycle regulation, and DNA repair pathway choice play major roles in determining the editing outcome. Therefore, genome editing approaches have begun to harness DNA repair control and modulation for more efficient and predictable outcomes.ex vivo editing strategies allow for extensive quality control, in vivo editing strategies could target multiple cell types at once, but must be safe and accurate, especially when targeting long-lived somatic stem cells. Recent successes in therapeutic editing achieved in β-thalassemia and sickle cell anemia demonstrated the feasibility of utilising CRISPR-Cas9 editing in stem cells to alleviate disease. While these reports are encouraging, there is a large margin for improving treatment strategies for diseases which require editing of multiple loci or precise editing of one locus across multiple tissues. CRISPR technologies that do not rely on the generation of DSBs, such as DNA base editors and prime editing, are promising avenues for future precision medicine. These technologies are independent of cell cycle stage and hence have the potential to correct multiple cell types. However, both base editors and prime editing introduce unique types of DNA damage products, such as DNA single-strand breaks and base mismatches, to facilitate genome editing. Hence these approaches rely on other DNA repair pathways that must be understood, in tissue-specific contexts, for further expansion and improvement of these technologies (Overall, the genome and transcriptome of target cells impact the effectiveness of genome editing approaches. Moreover, cell identity and tissue context are important considerations in designing effective editing strategies. While nologies .The expansion of the tools available to understand and control the CRISPR-Cas9 system has continuously fuelled the development of new therapeutic strategies and has brought a fundamental discovery into the clinics in less than a decade. The implications for personalised medicine are immense. However, for this steep trajectory to continue and to broaden the applicability and impact of these technologies, the focus of future developments must shift to include the investigation of tissue specific DNA repair. Knowledge of the underlying mechanisms of how the DNA repair machinery reacts to a CRISPR break within a distinct cellular context is a key to mapping the landscape of genome editing.MM and JF wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Telehealth delivery expanded quickly during the COVID-19 pandemic after the reduction of payment and regulatory barriers, but older adults are the least likely to benefit from this expansion. Little is known about physician experiences initiating telehealth and factors that fostered or discouraged adoption during the COVID-19 pandemic with older adult patients. Therefore, our objective was to understand experiences of frontline physicians caring for older adults via telehealth during the COVID-19 pandemic.n = 18), primary care (n = 15), and emergency (n = 15) physicians from all United Stated (US) regions, rural-urban settings, and academic-community practices who cared for older adult patients during the pandemic using purposive sampling methods. We completed framework analysis of the transcribed interviews to identify emerging themes and used the Quadruple Aim to organize themes.We conducted semi-structured interviews from September 2020 to November 2020 with 48 physicians. We recruited a diverse sample of geriatricians (Frontline physicians described telehealth as a more flexible, value-based, and patient-centered mode of health care delivery. Benefits of using telehealth to treat older adults included reducing deferred care and increasing timely care, improving efficiency for physicians, enhancing communication with caregivers and patients, reducing patient travel burdens, and facilitating health outreach and education. Challenges included unequal access for rural, older, or cognitively impaired patients. Physicians noted that payment parity with in-person visits, between video and telephone visits, and relaxation of restrictive regulations would enhance their ability to continue to offer telehealth.Frontline physicians who treated older adults during the COVID-19 pandemic were largely in favor of continuing telehealth use beyond the pandemic; however, they noted that sustainability would depend on enacting policies that address access inequities and reimbursement concerns. Our data provide policy insights that if placed into action could facilitate the long-term success of telehealth and encourage a more flexible healthcare delivery system in the US.The online version contains supplementary material available at 10.1186/s12877-022-02860-8. In the United States (US), the coronavirus disease COVID-19) pandemic promulgated a 154% increase in telehealth visits in March 2020 compared to March 2019 9 pandemi, and (3)Older adults (aged 65 years and older) are the most vulnerable to severe complications and death due to COVID-19 –10. WhilExpanded telehealth use has the potential to permanently transform healthcare delivery systems and access to care for older adult populations facing challenges to in-person care , as wellThe Quadruple Aim, a conceptual framework that optimizes health system performance, seeks to guide development of high value care for patients while encouraging a transition to population health through four domains: patient care experience, population health, healthcare costs, and workforce engagement and safety , 18. ExaWe conducted semi-structured virtual interviews with geriatricians, primary care, and emergency physicians from diverse settings. Telehealth was defined broadly as remote communication with patients, including telephone calls, audiovisual visits, home monitoring with wearables , web portals, or app-based communication. The principal investigator’s hospital’s Institutional Review Board (IRB) approved the study. We follow the Consolidated Criteria for Reporting Qualitative Research (COREQ)  in reporStudy eligible physicians were licensed to practice in the US in geriatrics, primary care , or emergency medicine (EM). Because we sought to understand physician experiences caring for older patients, given their risk for COVID-19 isolation and morbidity, participants must have cared for patients 65 years and older in-person or via telehealth during the pandemic.We stratified recruitment by specialty and practice type (academic and community) to overcome limitations of prior telehealth research that primarily included physicians from large academic health systems . Using pWe developed an interview guide which contained a description of study objectives and open-ended questions with probes for follow-up questions . The first author is a female physician with formal qualitative research training and was known to some participating physicians. The fourth author is a female graduate student with 3 years of professional experience in qualitative research techniques. After obtaining verbal consent, interviews were audio-recorded, transcribed, and de-identified. The interviewer completed a written debriefing after each interview to document findings such as new or emerging themes and additional questions that should be added. Participants received a gift card after completing the interviews. No repeat interviews were conducted. Participant feedback was not sought. No one refused or dropped out of interviews.framework analysis that involved summarizing content within categories into charts after transcription; this approach allows for the systematic analysis of participant perspectives and the incorporation new codes to a priori codes developed based on the study protocol  or really immobile patients into the office. But still be able to get the care that they need” .Physicians reported that telehealth enhanced access to low-barrier care by reducing deferred care and increasing timely care, reducing spread of communicable diseases, strengthening communication with caregivers, and facilitating health outreach and education, thus improving older patient health . Providing non-video telehealth appointments would address telehealth access concerns, as many patients could not participate in video visits due to lacking digital literacy, internet connectivity, or devices. For patients with mobility or transportation issues that make in-person visits undesirable, physicians noted that being patient-centered would mean allowing the patient to choose what mode of telehealth to use rather than restricting them to options that are reimbursed at a higher rate, such as video or in-person visits. For these reasons, some physicians practicing in a fee-for-service setting observed that widespread adoption of telehealth has the potential to transform healthcare financing and delivery, if sustained beyond the pandemic. One PCP noted that telehealth could “break the fee-for-service world” if progress made during the COVID-19 pandemic continued .In general, physicians in practices employing value-based models faced fewer barriers initiating telehealth and using patient-preferred telehealth modalities , which allowed for earlier or more seamless adoption of telehealth during the pandemic. Physicians also saw opportunities to provide innovative treatment models based in value and not volume, such as partnering with paramedic services to avoid unnecessary trips to the ED. Many physicians acknowledged that fee-for-service reimbursement models underappreciate the value of patient counseling, observation of clinical conditions, and time needed to address complex psychosocial patient concerns, at the expense of high-quality patient care. If telehealth visits were reimbursed adequately, physicians saw telehealth as a clinical strategy to ensure dedicated time to patient counseling and care continuity.Physicians expressed that reimbursement for telehealth should continue beyond the pandemic as current relaxed policies “assigned value” for previously uncompensated work . Multiple office-based physicians reported satisfaction with the temporary payment changes, with one physician stating, “I feel like I was already doing all this work, but not getting reimbursed … I’m so much happier [now]” . Because of this, many physicians reported a desire to continue providing telehealth visits as a supplement to their in-person practice beyond the pandemic  can help align organizational financial incentives with patients’ needs and providers’ professional ethics.Study participants across specialties and care settings detailed the unique ways in which the flexibility of telehealth during the COVID-19 pandemic improved patient care and their own professional satisfaction. Specifically, long-term payment parity for telehealth services relative to in-person care was repeatedly cited by participants as a requisite for long-term adoption. Of note, participants already engaged in alternative payment models, including accountable care organizations, Veterans’ Affairs, and Kaiser Permanente, experienced the most seamless adoption of telehealth. Future alternative payment designs could consider investments into telehealth infrastructure in population health payments, with higher payments to providers caring for patients with disproportionately lower access to telehealth services. Taken together, these findings suggest that the marked growth in telehealth adoption during the pandemic has provided a much-needed catalyst to address pre-existing challenges and accelerate the trend toward alternative payment models. Further research is needed to investigate the impact of low-cost services such as telehealth on overall healthcare utilization.Yet, while the overall experience of study participants with telehealth during the COVID-19 pandemic was largely positive, several challenges were noted which highlight other longstanding barriers to care delivery in the US. The concerns about telehealth access among marginalized patients and the challenges for the clinicians who disproportionately treat them is reminiscent of past concerns that even well-intended programs have the potential to widen disparities in healthcare access and outcomes . ParticuSeveral regulatory and policy changes are necessary to ensure the sustainability of telehealth. The temporary relaxation of state licensing requirements alleviated staffing shortages during the COVID-19 pandemic, both due to underlying variation in clinician density and due to differing impact of COVID-19 among US regions over time. The restoration of in-state licensing and credentialing may create unnecessary hurdles for providers seeking to provide telehealth care in the settings with the greatest shortages, such as rural communities , where tWhile we found positive impacts of telehealth on patient experience overall, additional research needs to be done to examine patient-centered outcomes under telehealth. While telehealth may increase efficiency in some settings, it may also lead to unintended adverse consequences such as increased specialist referrals. Many participants expressed concerns that telehealth could increase costs because they feared litigation related to misdiagnosis without a hands-on physical exam, suggesting need for further training on appropriate uses for telehealth with older patients. Future research should explore appropriate telehealth use and build the evidence-base on adapting in-person examinations for virtual settings.Our results reflect the experience of participant physicians from the first 6 months of the pandemic and might not be representative of its later phases. Physicians were from select specialties, and may not reflect experiences of physicians from other specialties or non-physicians. Patients and non-physician staff are important stakeholders in clinical practice transformation, and future research should explore their perspectives. Younger physicians with prior telehealth use may have been more likely to respond to our flyer. As such, more favorable views of telehealth may have been captured and results may not be generalizable to older physicians or those with less technology use. However, we found themes were similar among younger and older physicians in our sample. Although our study included only four (8%) rural physicians, in 2000 only 9% of US physicians practiced in rural areas . We did In summary, participating physicians largely described telehealth as having the capacity to reform healthcare delivery to be more flexible, value-based, and patient-centered. They identified several benefits and still existing challenges of telehealth relating to the Quadruple Aim and cautioned that the continued success of telehealth will depend on key policy and regulatory decisions. These physicians also expressed optimism and specific ideas of how telehealth adoption during the COVID-19 pandemic could catalyze healthcare delivery transformation in the years to come. Our data provide policy insights that, if placed into action, could facilitate the long-term success of telehealth and encourage a more flexible healthcare delivery system in the US.Additional file 1: Appendix 1. Interview Protocol. Appendix 2: Table 2. Themes and Representative Quotations from Interviews in the Study of Geriatricians, Primary and Emergency Care Physicians on Telehealth, 2020 (Domain: Care Experience). Appendix 3: Table 3. Themes and Representative Quotations from Interviews in the Study of Geriatricians, Primary and Emergency Care Physicians on Telehealth, 2020 . Appendix 4: Table 4. Themes and Representative Quotations from Interviews in the Study of Geriatricians, Primary and Emergency Care Physicians on Telehealth, 2020 (Domain: Cost). Appendix 5: Table 5. Themes and Representative Quotations from Interviews in the Study of Geriatricians, Primary and Emergency Care Physicians on Telehealth, 2020 (Domain: Workforce Engagement).


Obsessive compulsive disorder (OCD) can manifest as a debilitating disease with high degrees of co-morbidity as well as clinical and etiological heterogenity. However, the underlying pathophysiology is not clearly understood. Computational psychiatry is an emerging field in which behavior and its neural correlates are quantitatively analyzed and computational models are developed to improve understanding of disorders by comparing model predictions to observations. The aim is to more precisely understand psychiatric illnesses. Such computational and theoretical approaches may also enable more personalized treatments. Yet, these methodological approaches are not self-evident for clinicians with a traditional medical background. In this mini-review, we summarize a selection of computational OCD models and computational analysis frameworks, while also considering the model predictions from a perspective of possible personalized treatment. The reviewed computational approaches used dynamical systems frameworks or machine learning methods for modeling, analyzing and classifying patient data. Bayesian interpretations of probability for model selection were also included. The computational dissection of the underlying pathology is expected to narrow the explanatory gap between the phenomenological nosology and the neuropathophysiological background of this heterogeneous disorder. It may also contribute to develop biologically grounded and more informed dimensional taxonomies of psychopathology. In this review, we assessed the evolution of the computational modeling efforts that aim to study some aspects of obsessive compulsive disorder (OCD) pathophysiology. The computational and theoretical investigations support the move from the currently used nosological classification toward trans-dimensional approaches . This trSeveral computational approaches utilizing dynamical systems have been developed, which can provide mechanistic insights about pathological neural dynamics in OCD. In these modeling frameworks, coupled non-linear differential equation systems were manipulated and perturbed. The solutions of these non-linear dynamical systems can exhibit a steep attractor state , which can mimic states of perseveration, obsessions, and compulsions , 3, 46. Other computational studies found that changes in the excitatory and inhibitory balance pushes a cortico-striatal-thalamo-cortical (CSTC) pathway to states of generalized hyper-activity. Certain changes in global E/I and specifically in the local inhibition may trigger network oscillations and generate hyper-activity throughout the entire CSTC pathway in OCD , 6. ThisSeveral computational studies utilizing ML techniques investigated certain aspects of neuropathophysiology and symptom phenomenology by analyzing and classifying OCD patient data. We review some of them.A study using Random Forest decision trees found that clinically useful predictions of remission may not require an extensive battery of measures. A small set of assessments may efficiently distinguish between higher and lower risk OCD patients to inform clinical decision-making . RelevanTo identify brain regions relevant for OCD diagnosis, bagged linear SVMs were applied to structural MRI (sMRI) data for discrimination across 86 OCD patients and 86 control subjects. 39 brain regions were identified showing the largest differences between OCD patients and healthy controls and 36 of those were located in the frontal, temporal, and parietal cortices or in subcortical structures . A multiOCD is a heterogeneous disorder with varied symptom presentations, each of which may relate to distinct neuropsychological features. Traditionally, this heterogeneity was approached by using a symptom-based evaluation, but an alternative can involve focusing on underlying symptom motivations . Note thWith unsupervised learning, a combination of Principle Component Analysis (PCA) and a K-means clustering algorithm was utilized to separate subgroups in the compulsive-impulsive dimensions. Clustering converged to yield four subgroups: low compulsivity–low impulsivity group; two groups showing roughly equal clinical severity, but with opposing dimensions ; and the fourth with both high compulsivity and impulsivity and recording the highest clinical severity. The largest cluster of individuals with OCD was characterized by high impulsivity and low compulsivity . The ideA recent study based on multi-level brain imaging and behavioral data from children using the Random Forest classification algorithm identified four new brain-behavior groups cutting across neurodevelopmental disorders such as autism spectrum disorder, OCD, and attention-deficit/hyperactivity disorder . It was Another class of models were developed to simulate goal-directed behavior, where OCD patients may have impairment. Deficits in goal-directed control implies vulnerability for developing rigid habits . These mModel-based RL learns to represent the environment for goal-directed predictions and allows learning to guide actions most accurately, at the expense of high computational and energy costs. Model-free RL optimizes dynamics and heuristics for habit learning without external representations and it demands less computational and memory resources, but is inflexible and generalizes poorly .In healthy cohorts, individual differences in model-based learning predicted sensitivity to outcome devaluation, suggesting that an associative mechanism underlies a bias toward habit formation. But no evidence was found of a causal relationship between model-free learning and devaluation sensitivity .Most previous work focused on distinguishing between only two RL systems: model-based and model-free RL , as protModel-based (over model-free) strategies were found to be positively correlated with gray matter volume in the ventromedial prefrontal cortex (PFC) and caudate, regions that are critical for goal-directed control , 17. DysIn adults, stimulant addiction and OCD were associated with a significant shift in habit formation and this abnormality can be quantified as model-free learning. Lower gray matter volumes in the caudate, medial OFC, and lateral prefrontal cortices were associated with a greater shift toward model-free habit formation .A plethora of studies have built on the idea that the brain implements Bayesian inference. This can be formalized in a Bayesian state-space model that aims to infer the current state of the environment by combining prior knowledge and current evidence, weighting each by its relative uncertainty. With this, learning is governed by the balance between uncertainty on state transitions and observational uncertainty .Some theoretical works analyzed the assumption that OCD patients have excessive uncertainty regarding state transitions. In this case, high transition uncertainty results in increased relative weighting of prediction errors. This could explain findings of increased responses to predictable stimuli. The increased weighting of prediction errors seems more likely to be the result of high transition uncertainty than underestimation of sensory noise. Increased weighting of prediction errors are related to perceiving the world as more unstable. Further, the above alterations could account for sensory over-responsiveness in OCD, as well as the experience of intrusive thoughts. Overweighting of sensory data often implies an impairment in processing, as it leads to a failure of the use of prior information and less attenuation of sensory noise. As a further consequence, this can manifest in patients' experiences that actions were not performed correctly, obsessional thoughts, compulsions, and sensory over-responsiveness , 39.Severe cognitive flexibility impairments in OCD have been described in several studies , althougIn a combined experimental and computational study, it was shown that OCD patients develop an accurate internal model of the environment but they use it less to guide behavior. This suggests a cognitive architecture that separately interprets the environment independently of performance . DiffereEnhanced activation in the fronto-cingulate system in OCD patients and task-related modulation of effective connectivity from the dorsal anterior cingulate cortex (ACC) to left dorsolateral PFC was demonstrated by using dynamical causal modeling (DCM) on patient fMRI data. These findings implicated an overactive error control system in OCD . AnotherPatient insight in OCD is crucial. The diagnostic status of poor insight is ambiguous but is a key clinical factor that influences therapy outcome . Poor inOCD patients with good and poor insight (OCD-GI and OCD-PI) have partly distinct brain structural alterations . OCD-PI Trans-dimensional biologically grounded approaches to OCD symptoms are supported by the obvious existence of OCD sub-types. A shift from a categorized disease framework to a dimensional one may enable more personalized treatment choices . We listD2/3 agonists and antagonists had similar effects on OCD groups, as both increased punishment-driven learning  and OCD. Stimulus-bound perseveration is a measure of how a subject is responding to a repeated stimulus, irrespective of outcome. This measure was found to be significantly increased in SUD, but decreased in OCD, compared to controls. Individuals with SUD exhibited reduced reward-driven learning, while both the SUD and OCD groups showed increased learning from punishment. Dopamine receptor learning . In additagonist . Modelintagonist .Another aspect of behavioral microstructure was analyzed by 2-level factor modeling in OCD patients. This modeling study found that heterogeneous symptoms  reflect four underlying symptom dimensions with deviations from previous results .Obsessions and compulsions might independently contribute to the pathophysiology , 16. An The neuropathophysiology of co-morbid OCD and schizophrenia was examined in a phenomenological computational model . It was The clinical implications of certain computational results suggest possible development of personalized medicine to identify and optimize specific therapies for individual OCD patients. We list some of those efforts. Pre-treatment functional connectivity patterns within the default mode network and visual network significantly predicted the effect of cognitive behavioral therapy (CBT) and post-treatment OCD severity. These networks were stronger predictors than pre-treatment clinical scores . AbnormaTranscranial magnetic stimulation (TMS) has shown promise as an adjunct treatment for the symptoms of OCD . QuantitTherapy refractory OCD patients have benefited from deep brain stimulation (DBS). Optimal therapeutic results are associated with the activation of distinct fiber pathways. The stimulation of the right anterior middle frontal gyrus (DLPFC) has shown a positive response. Focused stimulation of specific fiber pathways, which allows stimulation with lower amplitudes, may be superior to activation of a wide array of pathways, typically associated with higher stimulation amplitudes . Closed-A recent meta-analysis using a Bayesian hierarchical model framework examined adverse effects of selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) treatments in pediatric OCD and anxiety disorders. It was found that compared with SNRIs, SSRIs are more likely to produce activation such as insomnia, irritability, hyperactivity, and impulsivity. The results suggested that although SSRIs are superior to SNRIs and the treatment of choice, for those patients who become activated on SSRIs, SNRIs might represent a good second choice given their reported efficacy and lower risk of activation .There are computational contributions that were not included in the current work. The contents of the obsessive-compulsive symptoms  were only partially explored. Computational work with brain histology was also excluded. We did not include comprehensive aspects of the developmental trajectories of the disease . Further reviews are necessary to follow and categorize this rapidly growing field.To summarize, we reviewed some of the computational modeling efforts which were developed to explain certain aspects of OCD pathophysiology and symptomology. These models span from mechanistic dynamical systems approaches, across ML techniques which aim to integrate and classify patient data , to include Bayesian model selection frameworks. We related the modeling evidence and results to diagnostic procedures, co-morbid states, and therapeutical consequences. In conclusion, computational psychiatry has powerful methods, which can arm psychiatrists with more quantitative tools . AlthougAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.KS was supported by ALF, RUFU, Märta and Nasvells Stiftelse research grants provided by the Uppsala University Hospital. Support for DS was provided by a joint grant from the John Templeton Foundation and the Fetzer Institute.The opinions expressed in this publication are those of the author(s) and do not necessarily reflect the views of the John Templeton Foundation or the Fetzer Institute.The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.