For much of 2021, allocating the scarce supply of Covid‐19 vaccines was the world's most pressing bioethical challenge, and similar challenges may recur for novel therapies and future vaccines. In the United States, the Centers for Disease Control and Prevention's Advisory Committee on Immunization Practices (ACIP) identified three fundamental ethical principles to guide the process: maximize benefits, promote justice, and mitigate health inequities. We argue that critical components of the recommended protocol were internally inconsistent with these principles. Specifically, the ACIP violated its principles by recommending overly broad health care worker priority in phase 1a, using being at least seventy‐five years of age as the only criterion to identify individuals at high risk of death from Covid‐19 during phase 1b, failing to recommend place‐based vaccine distribution, and implicitly endorsing first‐come, first‐served allocation. More rigorous empirical work and the development of a complete ethical framework that recognizes trade‐offs between principles may have prevented these mistakes and saved lives. After reviewing the proposed ethical principles for Covid‐19 vaccine allocation from the National Academy of Sciences, Engineering, and Medicine,The ACIP comprises a group of “medical and public health experts who develop recommendations on the use of vaccines in the civilian population of the United States” that, if adopted by the CDC director, are published as official recommendations of CDC and the Department of Health and Human Services.The first ethical misstep of the ACIP was an act of commission: it defined “health care personnel” so broadly as to leave many high‐risk persons in the community unvaccinated while vaccine doses went unused for months. When deciding who in the United States should have first access to the Covid‐19 vaccines, the ACIP assigned two groups equal priority: health care personnel (HCP) and residents of long‐term care facilities. These residents were a clear first choice to maximize benefits due to the combination of high infection risk in congregate living and the increased risk of severe postinfection outcomes because of advanced age and chronic medical conditions. From the perspective of promoting justice, residents of long‐term care facilities cannot protect themselves from infection by staff or visitors. Finally, prioritizing residents in these facilities may mitigate some racial health inequities: racial and ethnic minorities have access to lower‐quality long‐term care facilities and, prior to vaccine availability, suffered dramatically higher rates of nursing home Covid‐19 deaths than their White counterparts.In contrast, the ethical justification for the broad definition of HCP is less clear. The ACIP defined HCP as “all paid and unpaid persons serving in health care settings who have the potential for direct or indirect exposure to patients or infectious materials,” and health care organizations predictably operationalized this as anyone who works for a hospital or academic medical center.The argument for HCP priority rests primarily on the premise that “early protection of health care personnel is critical to preserve capacity to care for patients with Covid‐19 or other illnesses,”The ACIP asserted that broad health‐care‐worker priority was also supported by the increased occupational risk of Covid‐19. It is unclear whether this falls under the principle of promoting justice  or the principle of maximizing benefits (because vaccinating highly exposed people prevents more harm). Either way, the assertion is dubious. While health care workers in the United States have a higher occupational risk of Covid‐19 infection, most had substantially higher access to free personal protective equipment than did other essential workers, reducing their relative risk of infection. Perhaps for this reason, rates of excess mortality from the virus did not significantly differ between HCP and frontline essential workers in other fields.Finally, there are substantial racial and ethnic inequities in representation among higher‐income health care professionals (such as physicians and nurses).The ACIP protocol stands in stark contrast to the recommended jump‐start phase of first responders and high‐risk health care workers proposed by NASEM. Vaccinating a small group of workers essential to the pandemic response would have been consistent with the ACIP principles. Instead, hundreds of thousands of doses  sat idle in freezers in January 2021.under seventy‐five. Covid‐19 risk is a product of the probability of infection and death after infection. A wealthy seventy‐six‐year‐old able to live independently and shelter in place in their home is at substantially lower risk of Covid‐19 infection than a sixty‐six‐year‐old grandmother in a congregate living setting with multiple essential‐worker family members. Ignoring other medical risk factors also prioritizes individuals at lower risk over those at high medical risk. According to the protocol, a seventy‐three‐year‐old with diabetes and advanced congestive heart failure should receive lower priority for vaccination than a healthy seventy‐five‐year‐old.The second ethical error was using age as the single measure to determine eligibility for persons who were not frontline essential workers during phase 1b. In constructing phase 1b, the ACIP assigned equal priority to frontline essential workers and all persons seventy‐five years of age and older. The ACIP chose being over seventy‐five as a simple way to identify individuals at high medical risk, as about half of all U.S. Covid deaths prior to vaccine availability occurred in this age group.One‐size‐fits‐all age thresholds were often defended on the basis of simplicity. But other factors, such as place‐based risk, could also be incorporated easily using the same types of identification documents used to confirm age.A strict age cutoff also violates the justice principle. By prioritizing only those who have been fortunate enough to reach seventy‐five years of age, this cutoff disproportionately disadvantages those who have not had the opportunity to live to seventy‐five and would be unlikely to even without the threat of Covid‐19.Finally, the ACIP created significant structural inequities by race when they failed to account for the U.S. age distribution by race in this recommendation. The average age of a White person in the United States is fifty‐eight, more than twice the average age for racial and ethnic minorities.The third protocol error—one of omission—was the failure to consider place‐based risk and geographic variation in determining Covid‐19 vaccine distribution. Covid‐19 case rates, morbidity, and mortality have been unequally distributed across the United States since the start of the pandemic. In the first wave of the pandemic, death rates ranged from over eight hundred a day in New York to just one per day in Vermont.Hastings Center Report in the spring of 2020.Within major cities, Covid‐19 was distributed along predicted lines of socioeconomic status and other social‐vulnerability measures, exacerbating long‐standing disparities in health care and health outcomes. According to multiple studies, racial and ethnic minority groups were more likely to be exposed to Covid‐19 and suffer severe outcomes like hospitalization and death at younger ages if infected.Further research confirms that this act of omission dramatically reduced the potential number of lives saved, particularly among vulnerable populations, violating both the principle of maximizing benefits and the principle of mitigating inequities by not allocating vaccines proportional to risk.Some may argue that asking individuals at low geographic, or place‐based, risk to wait for vaccines is inconsistent with the equal‐opportunity dimensions of the ACIP's “promoting justice” principle.The final ethical error was another act of omission. This was the failure to specify an ethical mechanism for within‐phase vaccine distribution. While the ACIP protocol defined allocation phases based on groups prioritized for vaccination due to increased risk of coronavirus infection or mortality, the allocation phases themselves were massive, ranging from 24 million to 129 million persons, with significant heterogeneity of within‐group risk . The ACIP provided little direction for within‐phase allocation, aside from recommendations that local health authorities should “sub‐prioritize within an allocation phase if necessary.”First‐come, first‐served approaches (rank ordering patients by waiting time) lead to equal opportunity for maximum health and well‐being only when it is possible for people with equivalent expected benefit to have equal access to the waiting list when they develop the need for the resource. For example, since requiring dialysis for end‐stage renal disease dramatically reduces health and well‐being, a system that ranks candidates for kidney transplantation by the date on which they need to start dialysis treatment would lead to equal waiting time suffering on dialysis. However, there is no analogous waiting time in a global pandemic. When each priority phase was activated, millions of people were instantly newly eligible for the vaccine.A first‐come, first‐served approach prioritizes the well connected and well‐off, who have the luxury of time to wait in line, the financial resources to pay someone to wait for them, or the social capital to cut to the front of the line. While one could speculate that a first‐come, first‐served approach might select for those who are most desperate for a vaccine and further speculate that such people would benefit more than average, there is no evidence that this happened during Covid‐19 vaccine allocation. In reality, when combined with online‐only sign‐up systems, first‐come, first‐served vaccine allocation exacerbated health disparities , violating the principle of mitigating inequity.The ACIP should have strongly recommended randomized lottery systems to allocate vaccines within phases.There are consistent themes in the mistakes the ACIP made during protocol development. First, they did not engage empirical data properly when translating their ethics framework into recommendations. Rather than relying on intuitive speculation for many prioritization decisions (such as the use of age seventy‐five as a threshold for eligibility or the broad definition of HCP), the committee should have used more sophisticated models that simulate the dynamic spread of Covid‐19 in the U.S. population to quantify the potential lives saved by vaccinating members of each group.Second, although the ACIP listed three reasonable and acceptable ethical principles, the committee made no effort to rank order the principles or assign them relative weights. This approach implies that there are no trade‐offs in scarce‐resource allocation and that the chosen group during each phase was optimal according to all three principles. Yet vaccinating high‐risk adults would have saved more lives than would vaccinating just anyone loosely affiliated with a hospital. The ACIP did not acknowledge this tension, instead allowing the public to assume that HCP priority best satisfied all three principles.One might object that these criticisms are unfair because the ACIP's membership overwhelmingly comprises clinicians and public health professionals, who do not typically design scarce‐resource allocation protocols. However, they had access to consultants as well as NASEM's expert report. The ACIP could have engaged a consultant team with domain expertise in the ethical design of scarce‐resource‐allocation protocols, or could have simply adopted NASEM's recommendations. In contrast to the membership of the NASEM committee, no author of ACIP's allocation guidance, a group that comprised both the ACIP members and consultants, held a doctorate in bioethics, philosophy, or any social science. The majority held only MDs, and only two members had doctoral degrees in nonclinical subjects . To be sure, formal credentials are neither necessary nor sufficient to engage complex scarce‐resource‐allocation problems, but consulting with experts in the field might have helped the ACIP avoid its missteps.The task of converting ethical principles into a vaccine‐allocation protocol was an enormous bioethical challenge. It is critical for the bioethics community to acknowledge the shortcomings of the ACIP protocol, identify the root causes of the missteps, and build a better set of empirical bioethical tools to convert principles into protocols. Lives were, and remain, on the line.


Daphnia dentifera hosts were sampled from five Indiana lakes between June and December 2017 before and during epidemics of their fungal pathogen, Metschnikowia bicuspidata. Collected Daphnia were experimentally exposed to Metschnikowia and assayed for their gut penetrability, hemocyte response, and multi‐locus genotype. Mixed‐effects models were constructed to partition variance in immune traits between genetic and environmental sources. We then isolated the genetic sources to produce genotype‐specific estimates of immune traits for each multi‐locus genotype. Finally, we assessed the relative frequency and dynamics of genotypes during epidemics and asked whether genotypes with more robust immune responses increased in frequency during epidemics. Although genotype was an important source of variation for both gut penetrability and the hemocyte response, environmental factors  still explained a large portion of observed variation, suggesting a high degree of flexibility in Daphnia immune traits. Additionally, no significant associations were detected between a genotype's immune traits and its frequency in a population. Our study highlights the power of variance partitioning in understanding the factors driving variation in Daphnia traits and motivates further research on immunological flexibility and the ecological drivers of immune variation.Host immune traits arise from both genetic and environmental sources of variation. When immune traits have a strong genetic basis, the presence and severity of disease in a population may influence the distribution of those traits. Our study addressed how two immune‐related traits (gut penetrability and the hemocyte response) are shaped by genetic and environmental sources of variation, and how the presence of a virulent disease altered the relative frequency of these traits in natural populations.  Daphnia dentifera immune traits, but environmental factors were also important, suggesting a high degree of flexibility in their immune traits. We also found evidence that Daphnia population genetic structure does not shift toward more resistant genotypes during epidemics, which counters laboratory‐based evidence of rapid evolution of resistance and highlights the importance of considering the role of plasticity and the presence of multiple selection pressures when searching for evidence of natural selection.We found that genetics shape a large amount of variation in key  We then isolated the genetic sources to produce genotype‐specific estimates of immune traits and explored how each genotype's immune traits were associated with its abundance during epidemics. Given that many Daphnia traits are influenced by both genetics and the environment  occurring in the fall , Downing (n = 184), Hale (n = 158), Midland (n = 190), and Star (n = 163)). Field‐collected Daphnia were experimentally exposed to Metschnikowia and assayed for their gut penetrability, hemocyte response, and multi‐locus genotype.To analyze connections among disease dynamics, immunity traits, and clonal structure in l et al. , Rogalskl et al. , and Stel et al. . In brie2.2Metschnikowia spore will penetrate the gut barrier) is the first line of defense for Daphnia and was measured for each individual by recording the number of spores that entered the body cavity versus the number of spores that attacked the gut epithelium but failed to enter the body cavity  . We constructed generalized linear mixed models for each lake to partition variance in the two traits. In our models, the only main effect was the intercept, and we incorporated genotype, resource availability, MicG infection, and Metschnikowia abundance as categorical random effects. We then attributed variance to each random effect by dividing the variance of the random effect by the total model variance . Variance partitioning models were constructed using the glmmTMB package in R (version 1.1.1)  or environmental variation  were defined as attacking spores that succeeded in penetrating the gut epithelial tissue, and failures (“0”) were defined as attacking spores that failed to penetrate the tissue (successes and failures were incorporated into the response variable using “cbind” in R). By considering the number of successes and failures for each 2.4.2Our next question was whether epidemics shaped the relative abundance of genotypes depending on their susceptibility. To address this question, we first needed to isolate the genetic signal from the environmental noise. In other words, given that environmental conditions could influence the expression of immune traits, we sought a means to control for that environmental variation and extract genotype‐specific estimates of immune traits. To generate genotype‐specific estimates of gut penetrability and hemocyte responses, we constructed the same generalized linear mixed models as above , and both possessed relatively impenetrable guts. Without successful spore penetration, spores with hemocytes in the body cavity do not exist, such that we could not model the hemocyte response.We ran the models (above) for only frequently observed genotypes. By focusing on genotypes with larger representative sample sizes, we aimed to increase precision in estimating genotype‐specific immune traits and increase the likelihood that we would observe any changes in a genotype's abundance over time  by dividing the number of individuals representing each genotype by the total number of genotyped individuals in the lake sampled on a given date. We arcsin transformed these frequencies because they represent proportional data. The epidemic timeframe from which relative frequency values were obtained ranged from the sampling date immediately prior to epidemic emergence (when the exposures leading to infection in the epidemic first occurred in each lake) through the course of the epidemic until sampling ceased  shaped gut penetrability . However, there was no significant difference in gut penetrability among the two genotypes in Midland Lake . Within each of the lakes (with the exception of Midland which was excluded), there was significant variation in hemocyte responses among genotypes . Hemocyte responses ranged from 0 to 6 hemocytes per penetrating spore.Genotype‐specific gut penetrability ranged from highly impenetrable (near‐zero in Midland Lake) to moderately penetrable . This suggests that the relative abundance of each genotype was not related to its gut penetrability or its hemocyte response. We also detected no significant interaction effect between genotype‐specific immune traits and sampling event, suggesting that the change in abundance of a genotype during an epidemic is not related to its susceptibility to infection .We did not detect any significant associations between immune traits and the relative frequency of genotypes during epidemics. We observed no significant associations between each genotype's relative abundance during the epidemic and immune traits , and environmental risk of infection (Metschnikowia abundance). In many cases, these environmental variables played a strong role in shaping Daphnia immune traits. Ultimately, we did not observe associations between patterns of clonal abundance and genotype‐specific immune traits. This may be because disease did not play an especially strong selective role (and other selection pressures may have acted simultaneously) or because the flexibility of the immune traits reduced each genotype's response to selection.Disease can play a strong role in shaping population genetic structure  have been applied to invertebrate immune traits with similar findings  was mostly dependent on environmental conditions, although a strong genetic signal was present , whereas the hemocyte response was predominantly shaped by resource quantity , this factor was less influential in the other three lakes. In those three lakes , resource availability played a stronger role in shaping gut penetrability. In alignment with Rogalski et al. , as well as establishing genetic markers for immune traits, would allow for inclusion of less represented genotypes in future studies.By taking the knowledge from laboratory studies about how environmental factors may affect Daphnia's susceptibility to infection with Metschnikowia. Our study raises several exciting questions. How does genotype shape immune responses in diverse ecological conditions? How plastic are Daphnia immune phenotypes? And is the selection pressure posed by disease strong or weak, or obscured by plasticity and other selective pressures present in the environment? Addressing the plasticity of Daphnia immune traits, and how genotype‐by‐environment interactions shape Daphnia immunity, will help us further understand the extent to which population genetic structure is shaped by disease.In conclusion, our results suggest that both genetic and environmental variation are important for shaping Grace H. Westphal: Conceptualization (lead); formal analysis (lead); visualization (lead); writing – original draft (lead). Tara E. Stewart Merrill: Conceptualization (supporting); data curation (lead); formal analysis (supporting); writing – review and editing (lead).The authors declare no competing interests.Appendix S1:Click here for additional data file.


To the Editor, The study builds on the evidence that T. cordifolia causes drug‐induced liver injury (DILI) and in some cases is associated with autoantibody production and probable drug‐induced autoimmune hepatitis (DI‐AIH). The authors should be complimented for their detailed analysis of their patients.I read with interest the article Tinospora cordifolia (Giloy)‐Induced Liver Injury During the COVID‐19 Pandemic—Multicenter Nationwide Study From India, by Kulkarni and colleagues.1 The  and is preferred by experts in DILI. Liver biopsy is one of the elements in the simplified scoring system required for a “definite” diagnosis of DI‐AIH, so it will be interesting to know what proportion of patients in the Kulkarni et al. series fulfilled scores of >6 (probable) in those without histology and what proportion scored ≥7 (definite) on the Simplified Diagnostic Criteria. This is important, because the field of DI‐AIH is muddled with information from case reports or series where patients fulfill only partial criteria for a diagnosis of DI‐AIH (such as presence of antinuclear antibody), including a lack of liver biopsy and a range of common immunological blood tests or levels of immunoglobulin G.The authors report the presence of autoantibodies in 60% of 43 cases, yet a diagnosis of probable DI‐AIH was established only in 18% (four of 22 patients with liver biopsy) based on the International Autoimmune Hepatitis Group's revised original score. This score is meant for research purposes and may not be ideally suited for DI‐AIH given the negative points allocated to those with exposure to hepatotoxic drugs. The simplified criteria for diagnosis of AIH was established for clinical cases3 and  and was noted in a dilution of 1:40 in 35% of the normal population.Furthermore, presence of autoantibodies is not specific for autoimmune disease; antinuclear antibodies are seen in 21% of alcohol‐related liver disease and 34% of nonalcoholic fatty liver disease4 and de novo AIH in a setting of underlying liver disease, not DI‐AIH.The distinctive feature that separates DI‐AIH from classical AIH is that of absence of relapse in patients with DI‐AIH or progression to cirrhosis, whereas with classical AIH, relapse is almost universal. In Kulkarni and colleague's series, details of whether or not and how many were treated with steroids or immunosuppression are lacking. This information is essential, along with how many achieved remission following corticosteroid/immunosuppression withdrawal, thus qualifying for a bona fide diagnosis of DI‐AIH. The proportion that relapsed following discontinuation and/or treatment with steroids would point toward giloy acting as a trigger of T. cordifolia‐containing medications add to the growing list of newer medicinal agents, such as infliximab, adalimumab, and etanercept, but more importantly highlight the important and surreptitious role of botanical agents in causing DILI in general and DI‐AIH in particular.Increasing reports of Nothing to report.


Microdeletions are associated with different forms of epilepsy but show incomplete penetrance, which is not well understood. We aimed to assess whether unmasked variants or double CNVs could explain incomplete penetrance.We analyzed copy number variants (CNVs) in 603 patients with four different subgroups of epilepsy and 945 controls. CNVs were called from genotypes and validated on whole‐genome (WGS) or whole‐exome sequences (WES). CNV burden difference between patients and controls was obtained by fitting a logistic regression. CNV burden was assessed for small and large (>1 Mb) deletions and duplications and for deletions overlapping different gene sets.Large deletions were enriched in genetic generalized epilepsies (GGE) compared to controls. We also found enrichment of deletions in epilepsy genes and hotspots for GGE. We did not find truncating or functional variants that could have been unmasked by the deletions. We observed a double CNV hit in two patients. One patient also carried a de novo deletion in the 22q11.2 hotspot.We could corroborate previous findings of an enrichment of large microdeletions and deletions in epilepsy genes in GGE. We could also replicate that microdeletions show incomplete penetrance. However, we could not validate the hypothesis of unmasked variants nor the hypothesis of double CNVs to explain the incomplete penetrance. We found a de novo CNV on 22q11.2 that could be of interest. We also observed GGE families carrying a deletion on 15q13.3 hotspot that could be investigated in the Quebec founder population. Epilepsy has a prevalence of ~3% and a high socio‐economic burden.Copy number variants (CNVs) are implicated in the etiology of epilepsy, especially in developmental epileptic encephalopathies (DEE) and genetic generalized epilepsies (GGE).The genetic mechanisms by which these CNVs could cause epilepsy or other developmental disorders remain unclear. In the case of microdeletions, several mechanisms have been proposed to explain their incomplete penetrance, including the unmasking of a recessive allele,Here, we investigated CNVs as well as deletions in different sets of genes. The burden of CNVs was assessed in individuals with epilepsy, their unaffected family members and population controls using whole‐genome genotyping data. The patients and controls were mostly derived from the Quebec founder population.This study was approved by the CHUM Research Center (CRCHUM) ethics committee and by the University of Quebec in Chicoutimi ethics board. Written informed consent was obtained from all patients  and adult controls.The epilepsy cohort was composed of extended families comprising affected and unaffected individuals with GGE or NAFE as well as DEE trios with unaffected parents previously collected in CHUM Research Center and CHU Ste‐Justine in Montreal and in the Hospital for Sick Children in Toronto as part of the Canadian Epilepsy Network (CENet) and diagnosed by neurologists. The clinical epilepsy phenotype was classified according to the current classification by the International League against Epilepsy (ILAE).Samples were processed on either the Illumina Omni Express  or the Illumina Omni 2.5 . Genotypes of all samples were merged and only positions present on both chips were kept. We further removed SNVs with more than 2% missing sites over all individuals and with HWE p‐value <0.001 using PLINK softwareA file was generated by the Genome Quebec Innovation Center in Montreal for each genotyped sample including Log‐R ratio (LRR) and B allele frequency (BAF) for all SNVs. PennCNV softwareWe only considered rare CNVs (<=1%) for further analyses. There were 2698 such CNVs in our dataset Table . The CNVCNVs were validated using either whole‐genome (WGS) or whole‐exome (WES) depending on the availability of such sequences and/or segregation in the family. For segregation, CNVs were considered as being the same if they overlapped at least 50%. Duplications and deletions were considered separately. Detailed sequencing methods for WGS and WES are described in Moreau et al.PennCNV was used to determine if CNVs were spanning genes (hg19). A CNV was considered to be in the coding region if it overlapped at least 80% of a gene. We also identified 152 genes that were previously associated with epilepsyhttps://cran.r‐project.org/web/packages/MESS/index.html) to account for familial relationships. The familial relationships were obtained using PLINK –genome option after pruning. For all burden analyses, odds ratios, 95% confidence intervals (CIs), and significance were calculated by taking the exponential of the logistic regression coefficients. We removed the unaffected DEE parents from the DEE burden analyses. Bonferroni multiple‐testing was calculated for 16 tests for both groups of analyses and threshold for significance was 0.003.We measured CNV burden for all epilepsy phenotypes for small and large (>1 Mb) rare deletions and duplications separately to evaluate relative contribution on epilepsy type risk. We also looked at rare deletions overlapping genes, epilepsy‐associated genes, genes with pLi >0.99 and known epilepsy hotspots Table . To assehttps://gatk.broadinstitute.org/hc/en‐us). The vcf file was recalibrated and filtered following the GATK best practice guidelines. SnpEff and SnpSiftSNVs in microdeletions were called WGS or WES. We performed joint calling of gvcf files that were merged into a single vcf file using GATK version 3.7‐0  against controls  and validated by segregation. The second patient had one duplication followed immediately by a 13 Mb deletion on chromosome 18 (chr18:63151948‐64412293dup and chr18:64525217‐77553173del), both validated by WES, but with no family information.CHRNA7 and has been reported previously in GGE patientsIn the present work, we found an excess of deletions of more than 1 Mb in GGE patients compared to controls, and to a lesser extent, in individuals from mixed families, comparable to previous findings.The only de novo large deletion was found in a patient from a mixed family , neurodevelopmental disorders, and intellectual disability,Another hypothesis that has been proposed to explain incomplete penetrance is the double CNV hit hypothesis.In conclusion, we found an excess of large deletions in GGE patients compared to unaffected familial controls from the CENet cohort and population controls from the Quebec Reference Sample and also compared the number of duplications in GGE patients. Most of the deletions are located at genomic hotspots in GGE, especially at the 15q13.3 site which could have been brought and disseminate by an ancestor of the Quebec founder population. We also found one de novo deletion that could explain the patient's phenotype and be of interest for the medical follow‐up. We could not find evidence of deleterious or regulatory variants on the homologous chromosome that would explain the incomplete penetrance of the disease among individuals having large deletions. The double CNV hypothesis could not be supported neither although we found two large CNVs in two NAFE patients including one deletion of 13 Mb that could be of interest for the patient and the clinician. We found missense variants within the deletion regions that seem not sufficient to explain the disease. Therefore, we think that there might be other genomic or epigenomic causes in addition to large deletions that would explain the incomplete penetrance of epilepsy‐related microdeletions, although we need more sequencing data to validate these findings.All authors participated in the study design and reviewed the manuscript. CM, FT, and AG performed the analyses. CM wrote the manuscript. SLG supervised the analyses. CL built and manages the SLSJ family cohort which provided the Saguenay population control samples.The authors declare no conflict of interest.Figure S1 LRR's PCA before (left panel) and after (right panel) PC correction. Symbols represent the different batches and colors of the different plates (many plates were sent for genotyping within one batch).Figure S2. Pedigree of the mixed patient (DEE in a NAFE family) carrying the de novo deletion. Unaffected individuals are in black.Click here for additional data file.Table S1 Rare CNVs in epileptic patients and controls.Click here for additional data file.Table S2 Recurrent deletions' description from Watson et al.Click here for additional data file.Table S3 Deletions in epilepsy genes.Click here for additional data file.Table S4 Deletions in epilepsy hotspots.Click here for additional data file.Table S5 Description of deletions >1 Mb.Click here for additional data file.Table S6 Unmasked missense variants.Click here for additional data file.


Magnaporthe grisea and Aspergillus oryzae. Especially, when genome co‐editing is applied to a target gene and a membrane transporter gene that aid the entry of toxic compounds into cells, the resulting clone can be categorized as an autotrophic and non‐genetically modified clone. Such a clone should easily apply to industrial fermentation without being restricted by a genetically modified status. Genome co‐editing will also be used to construct mutant strains with multiple target gene knockouts by eliminating multiple membrane transporter genes. This could substantially improve the productivities of valuable metabolites and enzymes in a stepwise manner. Thus, genome co‐editing is considered a potentially powerful method to knockout single or multiple target genes that can contribute to the breeding of filamentous fungal strains in the food industry.In most countries, genetically modified microorganisms are not approved for use for fermentation in the food industry. Therefore, random mutagenesis and subsequent screening are performed to improve the productivities of valuable metabolites and enzymes as well as other specific functions in an industrial microbial strain. In addition, targeted gene knockout is performed by genetic recombination using its enzyme genes as selectable markers to maintain self‐cloning status. However, random mutagenesis has a drawback as it does not guarantee improvement of the targeted function. Conversely, self‐cloning is rarely used to breed an industrial microbial strain. This is probably because a self‐cloning strain is similar to a genetically modified strain, as both undergo homologous recombination, although exogenous genes are not introduced. In this article, I discuss the usefulness of genome editing technology as a substitute for conventional techniques to breed filamentous fungal strains. This article particularly focusses on “genome co‐editing,” a genome editing technology used for knocking out two genes concomitantly, as reported in  Illustrative explanation of the key difference between genome editing of the target gene alone and genome co‐editing of both target and membrane transporter genes. Genome editing generated colonies all around the plate due to no selection pressure, while genome co‐editing generated single colonies due to selection pressure. Genome co‐editing can generate a knockout mutant of the target gene in both autotrophic and non‐genetically modified forms. In addition, 39 of these colonies (41%) were whitish in appearance, indicating that they harbored both wA and thiI knockouts  to form mature spores with detectable color . Forty plates were used for this experiment. At the end of the experiment, 104 single colonies were generated from 520 protoplasts; however, they were all greenish ; data curation (lead); investigation (lead); project administration (lead); resources (lead); visualization (lead); writing—original draft (lead); writing—review & editing (lead).None declared.None required.


A history of preeclampsia is associated with increased risk of coronary artery disease and experimental evidence suggests that a history of preeclampsia also increases the risk of restenosis. However, the extent to which a history of preeclampsia is associated with risk of restenosis after percutaneous coronary intervention in women is unknown.We included 6065 parous women aged ≤65 years with first percutaneous coronary intervention on 9452 segments 2006 to 2017, linking nationwide data on percutaneous coronary intervention and delivery history in Sweden. Main outcomes were clinical restenosis and target lesion revascularization within 2 years. We accounted for segment‐, procedure‐, and patient‐related potential predictors of restenosis in proportional hazards regression models. Restenosis occurred in 345 segments (3.7%) and target lesion revascularization was performed on 383 patients (6.3%). A history of preeclampsia was neither significantly associated with risk of restenosis  nor target lesion revascularization  compared with a normotensive pregnancy history. When term and preterm preeclampsia were investigated separately, segments in women with a history of term preeclampsia had a lower risk of restenosis . A history of preeclampsia was not significantly associated with death by any cause within 2 years of the index procedure .A history of preeclampsia was not associated with increased risk of restenosis but instead some evidence pointed to a decreased risk. To facilitate future studies and allow for replication, concomitant collection of data on pregnancy complication history and percutaneous coronary intervention outcomes in women is warranted. DESdrug‐eluting stentMBRSwedish Medical Birth RegistrySCAARSwedish registry for coronary angiography and angioplastySflt‐1soluble fms‐like tyrosine kinase‐1A history of preeclampsia was not associated with increased risk of restenosis following percutaneous coronary intervention in women aged ≤65 years.Some evidence pointed to a decreased risk of restenosis in women with a history of preeclampsia at term.There is insufficient evidence to clinically consider history of preeclampsia in women undergoing percutaneous coronary intervention.To facilitate future studies and allow for replication, concomitant collection of data on pregnancy complication history and percutaneous coronary intervention outcomes in women is warranted.Despite significant progress during the past 2 decades with the introduction of drug‐eluting stents (DES) and devices,In an experimental model, preeclampsia in mice — induced through overexpressing a soluble vascular endothelial growth factor receptor — resulted in amplified vascular remodeling following vascular injury after the postpartum period, with increased vascular smooth cell proliferation and vascular fibrotic response.We therefore hypothesized that women with a history of preeclampsia have higher risk of restenosis and need of TLR following PCI. To test this hypothesis, we conducted a large nationwide study by merging data of the patient's PCI procedures with their preeclampsia history, using data ascertained via comprehensive clinical registries in Sweden.We conducted a prospective cohort study linking data collected from 2 nationwide and comprehensive clinical registries in Sweden. The SCAAR (Swedish Registry for Coronary Angiography and Angioplasty) contains data from all Swedish centers performing PCI, whereas the MBR  contains data on the large majority of all deliveries in Sweden since 1973. As shown in Figure International Classification of Diseases, Eighth, Ninth, and Tenth Revisions . Diagnoses were set according to clinical practice at time of diagnosis and pre‐existing hypertension was also registered early in pregnancy. For further information on specific diagnoses, see Data Data on exposure in any pregnancy to preeclampsia or nonpreeclamptic hypertension in pregnancy were ascertained from the MBR. The register started in 1973 and includes data on almost all pregnancies leading to delivery in Sweden.Data on patient characteristics and peri‐procedural variables were ascertained from SCAAR, which contains data on all coronary angiography and PCI procedures performed in Sweden. The following variables were included in this study as procedural predictors of restenosis: indication for procedure, stented vessel, type of device(s) used, length of stent (if used), and stent diameter following procedure. We further included several patient‐related predictors for restenosis at the time of stenting: diabetes, smoking, hypertension, dyslipidemia, and previous myocardial infarction. To account for the general improvement of care during the study period, we also included a categorical variable indicating year of procedure .In the restenosis analysis the primary end point was clinical restenosis, which is routinely collected in SCAAR. We defined restenosis according to the definition in the SCAAR registry: a stenosis assessed by angiographic visual estimation (>50%) or by fractional flow reserve ≤0.80 in a previously stented segment identified by coronary angiography for any clinical indication performed anywhere in the country.The TLR was defined as either any repeat PCI on a segment targeted at the index procedure or CABG following the index PCI, whichever occurred first. New PCI was ascertained via the SCAAR registry and CABG procedures  via the Swedish in‐patient registry, a comprehensive national registry of inpatient care. To ensure that CABG surgeries already planned at the time of index procedure where not included as end points, we excluded patients with a secondary decision of CABG at index PCI as described above.To analyze the end point death by any cause, we defined the event of death as registered in the comprehensive Swedish cause of death register.P values are 2‐sided, P<0.05 was considered notable. All analyses were performed using SAS version 9.4.We performed 2 complementary sets of analyses, investigating both time to restenosis per‐segment and time to TLR per patient by history of preeclampsia. The former analyses allow for sophisticated consideration of peri‐procedural characteristics as the targeted segment is the unit of analysis, whereas the latter is the equivalent per‐patient analysis. We used Kaplan–Meier curves to plot time‐to‐event by history of preeclampsia and proportional hazard regression to estimate hazard ratios (HR) and account for plausible predictors of restenosis. The start of the study time in 2006 corresponds to when all covariables were routinely collected in SCAAR. All For the per‐segment analyses, we used Cox regression with a jackknife estimator of variance to account for the statistical dependence between coronary artery segments in the same patient. To account for all type of device combinations  and include them in 1 coherent analysis, we combined appropriate interaction terms of the 3 binary variables drug‐eluting, stent, and balloon. This allowed us to account for other stent‐related features  by including an interaction term with stent. All per‐segment models were censored at 2 years of follow‐up, end of follow‐up, death, or migration, whichever occurred first.For the per‐patient TLR analysis, we used standard proportional hazard regression and accounted for all predictors on a patient level. To use information on the segment level in the per‐patient analyses, we allowed for several possible alternatives within a single patient for the variable treated vessel . We also further accounted for the variable number of treated vessels in the TLR analysis. All models were censored at 2 years, end of follow‐up, death, or migration, whichever occurred first.For the mortality analysis, we used proportional hazards regression accounted for age and censored at 2 years, end of follow‐up, or migration, whichever came first. The small number of events prevented full consideration of the predictors included in the TLR analyses.We checked the proportional hazards assumption using graphical methods and tests of Schoenfeld residuals, which indicated no violations of model assumptions.We used multiple imputation to address missing covariable data on the patient level. We used multiple imputation by chained equations with 20 imputed data sets and estimates were combined by applying PROC MIANALYZE in SAS 9.4. As described above, patients with missing data on any segment‐level variable were excluded; 5.7% of the segments had missing data on at least 1 covariable. Data on preeclampsia exposure and end points were not imputed. For more information on missing data per variable see Table Our final sample consisted of 6065 patients with 9452 segments. Median time between first pregnancy and the index PCI procedure was 30.9 years . Patient‐ and procedural‐characteristics per segment, by obstetric history, are listed in Table During 16 084 segment‐years of follow‐up, 345 restenosis events occurred, translating to 2.1% restenosis rate per year of follow‐up and 3.7% absolute risk within 2 years. In Figure In the TLR (per‐patient) analysis 383 events occurred during 10 174 person‐years, translating to 3.8% TLR rate per year of follow‐up or 6.3% absolute risk within 2 years. In Table In the all‐cause mortality analysis 166 events occurred during 10 684 person‐years, translating to 1.55% rate of death per year. Neither history of preeclampsia  nor non‐preeclamptic hypertension in pregnancy  were significantly associated with death within 2 years from the index PCI.As an analytic validation, the univariable associations between the clinical predictors of restenosis and actual restenosis risk is presented in Table In contrast to our hypothesis, we did not observe a higher risk of neither restenosis nor need for TLR in women with a history of preeclampsia. Instead, we found some evidence indicating a lower risk of restenosis in women with a history of preeclampsia, with the rate of restenosis being substantially lower in coronary artery segments of patients with a history of term preeclampsia. However, this per‐segment finding did not translate into a dramatically lower risk of TLR in patients with a history of term (or any) preeclampsia, despite similar numbers of outcome events.According to the most widely accepted hypothesis, the cause of preeclampsia somewhat differs according to its debut during pregnancy.As our study is based on clinical registry data, the pathophysiological basis of why women with a history of preeclampsia would have lower risk of restenosis remains elusive. However, we hypothesize that this could be explained by a difference in endothelial progenitor cells by preeclampsia history. Based on the important role of endothelial progenitor cells in vascular healing,The clinical results in interventional cardiology are affected by the tradeoff between stent thrombosis, restenosis, and bleeding.The strengths of this study include the comprehensive study sample with data collected from nationwide clinical registries covering almost all deliveries and all PCIs performed in Sweden. This allowed us to consider a range of relevant procedural‐ and patient‐related predictors of restenosis in our analysis. Furthermore, we used multiple imputation to account for a small proportion of missing covariable data to avoid exclusion of otherwise eligible patients from the study. However, there are also limitations to our study that should be acknowledged. Firstly, the validity of our results is contingent on that there is no major residual difference in PCI‐related care  that is relevant for clinical restenosis by history of preeclampsia. We consider such difference to be unlikely as we observed no major difference in the restenosis estimates by preeclampsia history when we accounted for several important procedural‐ and patient‐related predictors. Secondly, our results mainly pertain to the younger age span of women undergoing PCI as we study women aged ≤65 years. We consider this age restriction necessary for study validity as the delivery registry started in the early 1970s, and older women to a much lesser extent therefore have complete preeclampsia history data available. Still, young‐ and middle‐aged women (aged <65 years) have increased risk of major adverse cardiovascular events following PCI compared with older women, which seems to partly be explained by a higher incidence of repeat revascularization.In conclusion, a history of preeclampsia was not associated with increased risk of restenosis after PCI but instead some evidence pointed to reduced risk. However, data on complete pregnancy complication history are seldom available in studies of women undergoing PCI. To facilitate future studies and allow for replication, concomitant collection of data on pregnancy complication history and PCI outcomes in women is warranted.This work was funded by the Swedish Research Council (2019–02082); Jeansson Foundations, Gothenburg, Sweden; Åke Wiberg Foundation, Stockholm, Sweden; The Swedish Heart‐Lung Foundation (20180312); Crafoord Foundation, Lund Sweden; The Swedish Society of Medicine, and research support from the health care authority in Region Skåne, Sweden .None.Data S1Tables S1–S5Click here for additional data file.


Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue‐specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large‐scale manufacturing of therapeutic‐grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell‐based therapeutics were discussed. Development of novel strategies and approaches for scaling‐up EV production and the quality control/quality analysis (QC/QA) of EV‐based therapeutics were recognized as the necessary milestones for future investigations. Although most of these trials were based on using endogenous (unmodified) EVs, the concept of using engineered EVs in clinical trials has lately gained attention in the treatment of cancer, familial hypercholesterolemia and COVID‐19  in September 2021. A pro versus con debate on whether EVs are ready for human therapeutic applications was presented by Dr. Eduardo Marbán and Dr. Phillip Yang. The advantages of EV‐based therapeutics, including biologic plausibility, immune privilege, specific targeting and engineering versatility and the ongoing clinical trials on utilizing EVs as therapeutics or diagnostics, were highlighted by Dr. Marbán Marban, . Three mTo address these challenges and identify milestones for future investigations in the next decade, the presentations and discussions between experts at this workshop were focused on the following four thematic sessions: (1) engineering the membrane of EVs for HLBS disease therapy; (2) engineering the cargo of EVs for HLBS disease therapy; (3) lessons from other cell membrane‐derived vesicles and other diseases and (4) leveraging EV biology for novel diagnostics and prognostics. At the conclusion of this workshop, the group of experts (expert panel) identified key areas for future studies required to improve the engineering of EVs for HLBS diseases, emphasized the need to develop novel techniques for precision analysis of EV‐therapeutic function and identified the technological milestones necessary for scaling‐up the production of therapeutic EVs.22+ channels to cells lacking these channels. The presence of fluorescent cargo on EVs allows size validation and characterization of EVs using new nano‐flow cytometry‐based or high‐resolution microscopy‐based approaches. These include total internal reflection fluorescence (TIRF) microscopy or super resolution Stimulated Emission Depletion Microscopy (STED) microscopy and their roles as complementary approaches was discussed. In addition to these methodologies for characterizing EVs, newer approaches designed to address limitations of current methods for EV isolation were also discussed. Specifically, use of size‐exclusion columns (SECs), high‐pressure liquid chromatography based‐asymmetric field flow fractionation (AF4), or magnetic beads‐targeted to cargo‐protein present on EV membranes were suggested to overcome the limitations of ultracentrifugation, which is known to lead to EV aggregation and contamination by soluble proteins and may not be practical to support commercial‐scale manufacture. Several areas of improvement were identified to engineer EV‐membranes for delivery of membrane proteins to target cells, such as (1) inclusion of poly‐ethylene‐glycol (PEG) in EV suspensions to improve fusion of EVs with recipient cells, (2) using cell or virus‐derived fusion proteins to ensure EV fusion to the target‐cell plasma membrane in vivo without promoting endosomal degradation of EVs following fusion, (3) elucidating the molecular mechanism of EV generation or secretion to increase the yield of EVs carrying specific membrane‐cargo and (4) developing strategies to enrich subsets of EVs carrying therapeutically potent levels of desired protein(s) on the membrane.The biochemical composition of the EV membrane and the repertoire of adhesive epitopes presented by the EV membrane dictate the efficacy of EVs for use in the treatment of HBLS diseases. During this workshop, several speakers discussed the advantages and limitations of existing strategies used for designing of EV membranes and several areas with room for further improvement were identified to guide the engineering of membranes for therapeutic EVs. Deborah Nelson from the University of Chicago introduced the concept of using EVs as vehicles for functional transfer of membrane proteins to target cells, such as engineering EVs to carry the chloride‐channel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein as a membrane cargo to CFTR‐deficient alveolar macrophages for restoring their phagosomal microbicidal activity or transferring the functional light‐activated ORAI‐1 CaIn the same session, Jennifer Lang from SUNY‐Buffalo highlighted several approaches for engineering EV membranes for potential application in cardiac cell repair post myocardial infarction (MI) or chronic coronary artery disease (CAD). Based on the existing evidence, EVs‐derived from cardiac progenitor cells, such as cardiosphere‐derived cells (CDC‐EVs), were suggested to be therapeutically beneficial in reducing cardiomyocyte apoptosis, leading to reduced infarct size in preclinical models of MI , which can be included on endogenous membrane proteins of EVs as a post‐translational glycosylation modification. The azide group allowed EV detection using fluorescence imaging and efficient EV immobilization in the extracellular matrix (ECM) using copper‐free click chemistry, which is based on the reaction of a dibenzocyclooctyne moiety (DBCO) present on collagen with the azide‐group present on EV surface. Data using mesenchymal stem cell (MSC)‐generated EVs was shared to confirm that the surface azide expression on EVs (Az‐EVs) had no effect on the EV‐size distribution, endothelial uptake of EVs, or EV‐dependent endothelial cell proliferation and migration in in vitro cell culture studies. Importantly, Az‐EVs were shown to exhibit stable retention within collagen matrix expressing DBCO and be more efficient than unmodified EVs even at lower doses in promoting angiogenesis and macrophage infiltration in DBCO‐modified hydrogels subcutaneously implanted in mice  in rodent models. Pulmonary vascular endothelial cells (PVECs) and pulmonary artery smooth muscle cells (PASMCs) are two cell types in the lung that are critical to maintaining vascular homeostasis; derangement of their intercellular communication is a key step in the pathogenesis of pulmonary vascular structural remodelling in all forms of PH . This strategy involves neutrophil disruption by nitrogen cavitation followed by separation of membrane components using series of ultracentrifugation steps and extrusion through a membrane of specific pore size (∼50–200 nm) to generate neutrophil‐membrane‐derived nanovesicles , carry a negatively charged membrane  and glycocalyx), lack organelles and contain a limited number of signalling proteins and small RNAs, such as miR‐451 in the cytosol ‐derived EVs, how they contribute to cell‐cell communication and how they can potentially be used in HLBS diseases therapy. Besides electron microscopy, newer techniques like nanoflow cytometry, super‐resolution fluorescence microscopy, dark‐field microscopy and magnetic levitation relying on presence of specific antigens on EVs were described as major approaches available for RBC‐derived EV analysis or identification of EV‐cargo, such as miRNAs in both high and low resource settings  was presented with arrays of tortuous channels functionalized with antibodies targeted against epitopes  expressed on EVs of interest  needed for microfluidic‐based EV isolation allows this approach to be used for biomarker discovery in small volumes of plasma from paediatric medulloblastoma patients. Preliminary evidence was also provided to show how EVHB‐Chip can be functionalized with ACE2 receptor for SARS‐Cov2 virus or cell‐specific Ab cocktail  to capture virus or cell‐specific EVs from the plasma of COVID‐19 patients, and how these EVs can be used for RNAseq to potentially identify patients suitable for immunotherapy or diagnosis of COVID‐19 based on detection of viral RNA in EVs or plasma. Although the microfluidic systems seem to allow high purity isolation of EVs from small volume plasma samples, several areas for further research and development were identified by the expert panel, such as the need to combine with sophisticated hardware and software, select optimum Ab cocktails, address limitations associated with the usage of whole blood and investigate how haemolysis, activation of blood cells, coagulation during blood or plasma storage may affect the efficiency of EV isolation , or cause sudden cardiac death Cooper, . PreviouThe last speaker in this session, Joost P. G. Sluijter from the University Medical Centre Utrecht Regenerative Medicine Centre, highlighted the therapeutic roles of EVs in cardiac tissue repair. Dr. Sluijter shared published and unpublished findings to demonstrate how EVs are believed to contribute to the beneficial effects of stem cell therapy. The percutaneous intracoronary delivery of EVs was shown to protect the myocardium against ischemia‐reperfusion‐induced injury in both mouse and canine models , has served as an advisor for Neurodex and ShiftBio and an ad hoc consultant with NeuroTrauma Sciences, Kineticos, King Abdulaziz University and Burst Biologics, and has held research grants from AgriSciX, Yuvan Research, and Ionis Pharmaceuticals. The remaining authors declare no competing financial interests.


Holter analysis requires significant clinical resources to achieve a high‐quality diagnosis. This study sought to assess whether an artificial intelligence (AI)‐based Holter analysis platform using deep neural networks is noninferior to a conventional one used in clinical routine in detecting a major rhythm abnormality.P<0.001).A total of 1000 Holter (24‐hour) recordings were collected from 3 tertiary hospitals. Recordings were independently analyzed by cardiologists for the AI‐based platform and by electrophysiologists as part of clinical practice for the conventional platform. For each Holter, diagnostic performance was evaluated and compared through the analysis of the presence or absence of 5 predefined cardiac abnormalities: pauses, ventricular tachycardia, atrial fibrillation/flutter/tachycardia, high‐grade atrioventricular block, and high burden of premature ventricular complex (>10%). Analysis duration was monitored. The deep neural network–based platform was noninferior to the conventional one in its ability to detect a major rhythm abnormality. There were no statistically significant differences between AI‐based and classical platforms regarding the sensitivity and specificity to detect the predefined abnormalities except for atrial fibrillation and ventricular tachycardia . The AI‐based analysis was >25% faster than the conventional one (4.4 versus 6.0 minutes; These preliminary findings suggest that an AI‐based strategy for the analysis of Holter recordings is faster and at least as accurate as a conventional analysis by electrophysiologists. ECG Holter monitoring is a common tool used for the detection and characterization of cardiac rhythm abnormalities.Deep neural networks (DNNs) are an emerging diagnostic tool in cardiology.The data supporting the findings of this study may be made available from the corresponding author on reasonable request. This multicentric comparative study aimed principally to demonstrate the noninferiority of a DNN‐based Holter analysis solution compared with a conventional ambulatory ECG analysis platform in correctly detecting 5 major rhythm abnormalities. It secondarily aimed at comparing all performance indicators for both diagnosis procedures. This study was approved by the local ethics committee, and subjects gave informed consent.A total of 1000 consecutive 24‐hour Holter recordings performed in adults from January to December 2018 were collected in 3 tertiary care hospitals in the Paris area. Those recordings were collected from unselected routine patients seen in the ambulatory monitoring lab of the 3 hospitals and are therefore representative, in terms of arrhythmia prevalence, of the population on which Holter recordings are usually performed. When patients had >1 recording performed, only the first one was included. Holters of patients implanted with a pacemaker or a defibrillator were excluded. All recordings were performed using a 3‐lead Holter device , with a sampling rate of 200 Hz.The Cardiologs Holter analysis platform is a cloud‐based and DNN‐based platform Figure . CompareThe platform uses 2 different neural networks, one for wave detection and one dedicated to rhythm classification. The wave detector is a DNN with a U‐net architectureHolters were analyzed by experienced electrophysiologists with the conventional platform as part of normal clinical practice. The second analysis with the DNN‐based platform was performed by a second group of less experienced cardiologists (<5 years of experience after training), who were blinded to the results of the first analysis. Analysis time was recorded by each physician in both cases. The presence or absence of 5 prespecified arrhythmias was analyzed: pauses (≥2.5 seconds), AF, flutter, or tachycardia, combined into a same categoryThe ability to diagnose these 5 prespecified arrhythmias was assessed for each platform. The large number (1000) and relatively long duration (≥24 hours) of Holter recordings hindered expert electrophysiologists from reviewing the entire recording signal by hand, independently of any platform, in a timely manner. The reference diagnostic (ground truth) was therefore determined using the interpretation of both platforms. In case of discrepancy on any prespecified arrhythmia between both platforms, an expert electrophysiologist reviewed both analyses and adjudicated the rhythm. Because both platforms can miss abnormalities, only a combination of the two can be used to evaluate the quality of the analysis in a way fair to both platforms. While there is still a possibility for an abnormality to be missed by both platforms, thus resulting in an overestimation of the sensitivity of the platform, this method allows for a rigorous estimation of the differences of errors. In addition, 5% of randomly selected Holters without discrepancies were fully verified by one electrophysiologist who had the Holter analyses of both platforms available.t‐test. All comparative tests were 2‐sided with a significance level of 5%. Analyses were performed with SAS software .Quantitative variables were described by median , minimum and maximum because of the non‐Gaussian distributions. Categorical variables were described by number and percentage. The AI‐based method would be considered noninferior to the classical one for detection of major rhythm abnormalities if the lower bound of the one‐sided 97.5% CI of the difference between the detection rate of the 2 strategies was less than the noninferiority margin, which was set at 10%. Sensitivity, specificity, positive predictive value and negative predictive value for each major arrhythmia type with nonparametric 95% CI were also reported for both strategies and compared. The time needed for the analysis was compared between both platforms using a paired Student The defined major arrhythmias were present in 22.2% of the cases Figure .P=0.01) and ventricular tachycardia , while the classical analysis had slightly higher specificity for AF detection although of limited clinical relevance .The DNN‐based platform was noninferior to the conventional one in its ability to detect a major rhythm abnormality . The sensitivities and specificities Figure  and 2C fP<0.001)  versus 6.0 minutes  Figure .To the best of our knowledge, this is the largest study evaluating the performance of a DNN‐based platform for Holter analysis. We found that the DNN‐based platform was noninferior to the classical strategy in terms of overall diagnostic sensitivity and specificity, with the analysis duration being significantly shorter by 27% on average. The higher ventricular tachycardia and AF sensitivities of the DNN‐based platform were mostly attributable to premature ventricular contraction quadruplets and short episodes of AF being missed by the classical strategy. The lower specificity of DNN for AF was mostly attributable to rare episodes of sinus tachycardia being misclassified as AF by the DNN‐based platform. Importantly, there were no more significant false negatives compared with the classical strategy.One of the key advantages with DNN is that its performance typically improves with increasing number of recordings analyzed; hence, accuracy is likely to improve in the future, with a subsequent reduction in time needed for manual overread. This is important, as the considerable time needed for analyzing Holters is still a concern and represents a significant burden on limited clinical staff, potentially delaying important results and limiting the wider adoption of this test, especially for stroke prevention.Previous studies have shown superiority of DNN over standard algorithms for 12‐lead ECG analysis in specific arrhythmias such as AFWith regard to the actual use of AI in medical technologies, patients and physicians could be apprehensive about a partly automated tool that could make errors and the risk–benefit balance is an important consideration.A limitation of our study is that all readers were specialized cardiologists and it is possible that results may differ with other readers  or longer recordings (reader fatigue). Another limitation was related to the experience of the physicians, which was different in the group using each platform. Cardiologists overreading the DNN‐based platform received only a 1‐day training on this platform and were less experienced than the group of electrophysiologists who performed the overreading of the conventional platform analysis, with which they were familiar. This might overestimate the standard platform performance and underestimate the analysis time reduction observed with the DNN‐based platform compared with prior studies.In conclusion, our findings demonstrate that the use of a deep learning approach can help classify a broad range of distinct arrhythmias from ambulatory ECGs with high diagnostic performance. DNN‐based platforms hold promise to provide accurate, time‐efficient, and, ideally, fully automated Holter analysis in the future.The French National Institute of Health and Medical Research (INSERM).Dr Fiorina is a medical expert for Cardiologs. Dr Gardella, A. Plesse, and C. Henry are Cardiologs employees, and J. Li is a Cardiologs cofounder. Other authors have no disclosures to report.


To perform an updated systematic review of randomised controlled trials examining the efficacy of at‐home foot temperature monitoring in reducing the risk of a diabetes‐related foot ulcer (DFU).Systematic review performed according to Preferred Reporting Items for Systematic reviews and Meta‐Analyses guidelines. Risk‐of‐bias was assessed using version 2 of the Cochrane risk‐of‐bias tool. Meta‐analyses were performed using random effect models. Leave‐one‐out sensitivity analyses and a sub‐analysis excluding trials considered at high risk‐of‐bias assessed the consistency of the findings. The certainty of the evidence was assessed with GRADE.Five randomised controlled trials involving 772 participants meeting the International Working Group on the Diabetic Foot (IWGDF) risk category 2 or 3 were included. All trials reported instructing participants to measure skin temperature at‐home at six or more sites on each foot using a hand‐held infra‐red thermometer at least daily and reduce ambulatory activity in response to hotspots (temperature differences >2.2°C on two consecutive days between similar locations in both feet). One, one, and three trials were considered at low, moderate and high risk‐of‐bias, respectively. Participants allocated to at‐home foot temperature monitoring had a reduced risk of developing a DFU  compared to controls. Sensitivity and sub‐analyses suggested that the significance of this finding was consistent. The GRADE assessment suggested a low degree of certainty in the finding.At‐home daily foot temperature monitoring and reduction of ambulatory activity in response to hotspots reduce the risk of a DFU in moderate or high risk people with a low level of certainty. Secondary outcomes were minor and major amputations. Other outcomes collected were adherence to foot temperature monitoring, frequency of contacting the study nurse or podiatrist, and amount of ambulatory activity reductions in response to hotspots. Outcome data were extracted for the latest time point reported. Other data extracted included age, sex, body mass index (BMI), duration of diabetes, glycosylated haemoglobin levels (HbA1C), ankle‐brachial pressure index (ABPI), and loss to follow‐up.2.3Risk of bias of each included trial was assessed independently by two of three authors  using version 2 of the Cochrane risk‐of‐bias tool for randomised controlled trials.2.4p‐values <0.05 were considered significant. Statistical heterogeneity was assessed using the I2 statistic and interpreted as low (0%–49%), moderate (50%–74%), or high (75%–100%).Meta‐analyses were planned to be performed for any of the primary and secondary outcomes if data were reported in at least three trials. A sub‐analysis was also planned to exclude any studies deemed to be at high risk of bias.2.5https://gradepro.org/) to evaluate the risk of bias, inconsistency, indirectness, and imprecision of the combined trial evidence.The overall certainty  of the evidence was assessed according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) using the GRADEpro guideline development tool (GDT) . Only two trials reported amputation outcomes with no significant difference between groups , with low statistical heterogeneity between studies (I2 = 49%)  than the total included patients (n = 772).A summary of the GRADE certainty  of the evidence assessment is provided in Table 3.6Adherence to at‐home foot temperature monitoring by intervention participants was reported in variable ways in three trials4This updated meta‐analysis suggests that the daily at‐home foot temperature monitoring and activity reduction in response to hotspots significantly reduce the risk of a diabetes‐related foot ulcer amongst people at moderate or high risk (IWGDF risk category 2 or 3). This meta‐analysis included 304 more participants than prior meta‐analyses, almost doubling the numbers previously included.While the overall meta‐analysis suggests this kind of intervention halves the risk of foot ulcer development, the sub‐analysis showed that the pooled RR reduction in the two trials deemed to be at a lower risk of bias was 0.75 compared to 0.30 in the three higher risk of bias trials. This finding was considered to represent a serious inconsistency using the GRADE assessment. The most recent trial did find a significant reduction in the risk of developing a foot ulcer amongst the intervention group despite having the smallest relative reduction in this outcome of all the included trials. Over time, usual preventative care as provided to control groups, such as the production and use of offloading footwear, has advanced, which may in part explain the smaller effect sizes found in more recent trials.Interestingly, all the included trials used the TempTouch  to measure foot temperature.There is already a great burden of self‐monitoring placed on people with diabetes and thus a variety of forms of support may be needed for effective implementation of at‐home foot temperature monitoring. Motivational interviewing is a counselling method that has been used in a variety of populations to support healthy behaviours, such as physical activity.Measurement of foot temperature during clinical assessments is an alternative approach to at‐home monitoring that has been suggested, although this is not feasible to perform on a daily basis. A recent clinical trial tested the benefit of such foot temperature monitoring performed at monthly intervals at an outpatient clinic.A number of limitations of the included trials and this meta‐analysis should be acknowledged. The risk of bias of three of the included trials was considered to be high. Elements considered to be at risk of bias included the randomisation process, deviation from the intended intervention, measurement of the outcome, and selection of reported results. There was also heterogeneity in follow‐up time and in reporting patient characteristics. The GRADE assessment identified a low level of certainty in the findings. Lastly, the included trials all took place in Europe and the US, therefore the overall effectiveness of this intervention outside of these continents remains uncertain. Hence, further evaluation of temperature monitoring interventions in different climates and in different populations, particularly those at different risk for ulcers, is needed to evaluate its scalability more globally.In conclusion, this meta‐analysis provides promising but low‐certainty evidence that daily at‐home foot temperature monitoring and reduction of activity in response to hotspots are effective at reducing the risk of a diabetes‐related foot ulcer in at‐risk people. Effective, user‐friendly, and affordable intervention systems are needed for foot temperature monitoring and the necessary thresholds of action in response to identifying hotspots, for widespread adoption of this preventative intervention.Jonathan Golledge, Malindu Fernando, and Chanika Alahakoon extracted data; Peter A. Lazzarini, Chanika Alahakoon, and Jaap J. van Netten performed the risk‐of‐bias assessment; Malindu Fernando performed the statistical analysis; Jonathan Golledge wrote the manuscript; All authors provided critical revisions of the manuscript; Jonathan Golledge, Peter A. Lazzarini, and Sicco A. Bus handled funding and supervision.The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.https://publons.com/publon/10.1002/dmrr.3549.The peer review history for this article is available at Supplementary Information S1Click here for additional data file.


The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single‐stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA‐modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures. Proteins can be sequestered and released from artificial cells by attachment of DNA strands and using DNA strand displacement reactions. The DNA elements use a combined regulation mechanism of electrostatic interactions and specific sequence recognition. This provides control over rate of protein release, amount of protein released, and the multiplexity of release. Inspired by natural communication processes, scientists have succeeded in implementing signaling networks in various bottom‐up synthetic cell designs in order to mimic intra‐ and intercellular signaling, and study such basic signaling events in a highly controlled manner.2, 3 TCoacervates have been increasingly studied as bottom‐up artificial cell platforms, owing to their crowded nature and their potential to act as framework on which semi‐permeable membranes can be assembled.21, 22gradient‐trimethylene carbonate)‐poly‐(glutamic acid) ‐poly‐(caprolactone‐) Figure A and B.[) Figure C, D. Fur) Figure E.In order to create an attachment site for the UPT strand, short 12 nucleotide (nt) ssDNAs were site‐specifically conjugated via their 5′ end to three different proteins to act as a complementary, sequence‐selective and responsive handle; enhanced Yellow Fluorescent Protein (eYFP), Myoglobin (Mb) and Horseradish peroxidase (HRP) were modified via a strain‐promoted azide‐alkyne cycloaddition  resulted in complete protein release within 10 minutes Figure B and S8.In an attempt to replicate the competitive environment in natural cells where many different signaling molecules are present, two closely related releasing strands were designed . Upon addition to eYFP loaded coacervates, only the strand with a 100 % complementarity to the handle sequence was able to release eYFP.The DNA strand displacement concept provides a simplified, yet important and highly challenging first step, towards mimicking the cellular multiplex control over protein release.35, 36Finally, the DNA‐triggered transport of a protein from one population of artificial cells to another was shown. This was achieved by the DNA‐triggered release of eYFP which contained a His‐tag, known to specifically bind within artificial cells containing nitrilotriacetic acid (NTA) amylose.Here we have reported the controlled sequestration and release of proteins from artificial cells using DNA strand displacement reactions. This synthetic analogue to protein secretion provides an unrivalled level of programmable control over the transport of proteins across synthetic cell membranes. The DNA elements enable the regulation of electrostatic interactions between proteinaceous cargo and the coacervate core. Moreover, the specificity afforded by the DNA elements provides a fine degree of control over parameters such as the rate of release, the stoichiometric amount of material released, and the multiplexity of release. These results demonstrate that this coacervate‐based artificial cell system represents a versatile platform to establish protein‐based signal transduction for studying more complex, lifelike chemical communication pathways in synthetic cells.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


A dominant focus in the treatment of chronic pain in children and young people is placed on self‐management. Self‐management can be broadly defined as “an individual's ability to manage the symptoms, treatment, physical and psychological consequences, and lifestyle changes inherent in living with a chronic illness” (p. 178).Developing adequate self‐management skills is of particular importance in later childhood and adolescence (further referred to as young people). This is because, at this timepoint, individuals begin taking greater responsibility for self‐management of their chronic pain while parents typically reduce their involvement in managing young people's pain.Many evidence‐based treatment programmes for chronic pain promote the necessary skills to self‐manage pain episodes such as using relaxation, acceptance‐based approaches, problem‐solving, continued and consistent engagement with exercises, and medication adherence.This special issue collected four cutting‐edge articles which expand upon the existing knowledge concerning the impact of developmental challenges that young people and their parents may face with regard to engaging with effective self‐management strategies for managing their chronic pain. Articles within the special issue adopt a range of methodologies  across various key stages of young people's development, including the stage of emerging adulthood  may fluctuate over time. Much as the often quoted ‘children are not little adults’ statement highlights the distinctness of children from adults, it is critical to appreciate the unique and detailed nature of changes in development across adolescence and early adulthood when understanding and promoting effective self‐management processes in young people who live with pain.