Early antiretroviral therapy (ART) initiation  limits establishment of the HIV reservoir. However, the effect of early ART initiation on the long-term dynamics of the pool of infected cells remains unclear.In this longitudinal analysis, we included cisgender men who have sex with men (MSM) and transgender women (aged 18–54 years) at high risk for HIV infection, enrolled in the ongoing longitudinal MERLIN study in Peru between Oct 28, 2014, and Nov 8, 2018. Participants were eligible if they had been infected with HIV less than 90 days before enrolment, and if they had cryopreserved peripheral blood mononuclear cell (PBMC) samples. Participants were stratified into three groups on the basis of whether they initiated ART at 30 days or less (acute group), at 31–90 days (early group), or more than 24 weeks (deferred group) after the estimated date of detectable infection. PBMC samples were collected before ART initiation and longitudinally for up to 4 years on ART. The main outcomes were to establish the size of the HIV reservoir before ART initiation and to assess the effect of the timing of ART initiation on the decay of the HIV reservoir over 4 years follow-up. We quantified viral load, and isolated CD4 cells to quantify total HIV DNA, integrated HIV DNA and 2-long terminal repeat circles. Longitudinal analysis of active and inducible HIV reservoirs were measured by quantifying the frequency of CD4 cells producing multiply-spliced HIV RNA ex vivo and after in-vitro stimulation with a tat/rev induced limiting dilution assay (TILDA). A mixed-effects model from the time of ART initiation was used to measure longitudinal decays in viral loads and each HIV reservoir measure in each of the three groups.We included 56 participants in this analysis, all of whom were MSM: 15 were in the acute group, 19 were in the early group, and 22 were in the deferred group. Participants in all three groups had similar levels of all HIV reservoir markers before ART initiation. All participants, including those in the acute group, had a pool of transcriptionally silent HIV-infected cells before ART initiation, as indicated by a substantial increase in TILDA measures upon stimulation. Longitudinal analysis over 4 years of ART revealed a biphasic decay of all HIV persistence markers, with a rapid initial decline followed by a slower decay in all participants. During the first-phase decay, the half-lives of both total and integrated HIV DNA and TILDA measures were significantly shorter in the acute group than in the early and deferred groups. During the second-phase decay, HIV reservoir markers continued to decline only in participants in the acute group.Participants who initiated ART within 30 days or less of HIV infection showed a steeper and more sustained decay in HIV reservoir measures, suggesting long-term benefit of acute ART initiation on reservoir clearance.The US National Institutes of Health and the Canadian Institutes for Health Research. However, a small pool of long-lived, infected memory CD4 cells is unavoidably established during the first few weeks of HIV infection8 Initiating ART early  leads to a rapid decay in viraemia9 and drastically limits the size of the HIV reservoir.19 Nonetheless, early ART initiation does not prevent establishment of the reservoir, as indicated by the occurrence of viral rebound when treatment is interrupted, even in individuals who have initiated treatment at the earliest stages of HIV infection.21 Studies in non-human primates have revealed that a long-lived, replication-competent HIV reservoir can already be established in the first few days of Simian immunodeficiency virus infection, when plasma viraemia is not yet detectable.23 Although early ART initiation does not prevent establishment of the HIV reservoir, several observational studies have suggested that prompt ART initiation  might be associated with a more rapid decay in the pool of infected cells compared with delayed ART initiation ,19 which could accelerate reservoir clearance when combined with other eradication strategies. Several groups have described a biphasic decay of the HIV DNA reservoir on ART initiation, with a rapid initial decline followed by a second slower decay.26 Even though HIV DNA dynamics follow a similar two-phase decay pattern in early or chronically treated individuals, early ART initiation could be associated with a more rapid initial clearance of the HIV reservoir and a slower but continuous decay after long-term therapy compared with delayed ART initiation.27 Indeed, one study, published in 2020, in which an intact proviral DNA assay was used, revealed that intact proviruses might decay more rapidly than defective proviruses.28 However, the size of the inducible reservoir after long-term therapy has not yet been assessed in a large longitudinal study. Some studies have used the quantitative viral outgrowth assay to evaluate the dynamics of the functional HIV reservoir in a small number of participants.12 However this assay largely underestimates the size of the inducible HIV reservoir and has a narrow dynamic range. In contrast, assays that measure the frequency of CD4 cells with inducible RNAs, such as the tat/rev induced limiting dilution assay (TILDA),29 provide more sensitivity and a greater dynamic range for monitoring reductions in the HIV reservoir over time.During acute HIV infection , plasma viral load increases rapidly, peaking after 3–4 weeks before declining to a viral set point.The aim of this study was to assess the effects of the timing of ART initiation on the longitudinal persistence of HIV DNA and reservoir markers over 4 years of ART.NCT02744040) in Peru between Oct 28, 2014, and Nov 8, 2018.In this longitudinal analysis, we included cisgender men who have sex with men (MSM) and transgender women (aged 18–54 years) at high risk for HIV infection, enrolled in the ongoing longitudinal MERLIN study (NCT01815580),30 which enrolled 2109 cisgender MSM and transgender women (aged 18–73 years) at high risk for HIV infection in Lima, Peru, between Aug 5, 2013, and Sept 29, 2015. Participants were screened monthly for HIV RNA and serology to detect incident infections. HIV screening was done on whole blood using the third-generation HIV antibody test, Alere Determine HIV-1/2 . Blood samples negative for HIV antibodies were pooled and tested for HIV RNA with a nucleic acid amplification test . Positive pools were deconvoluted to identify HIV-positive samples using a fourth-generation HIV enzyme immune-assay  or HIV RNA tests. The Abbott RealTime HIV-1 Viral Load Assay  was used to confirm HIV infection. Participants were eligible for the Sabes study if they were seronegative but positive for HIV RNA (acute infection) or were seropositive less than 90 days from their last negative HIV RNA or serological test (early infection). 216 individuals who were diagnosed during acute and early HIV infection consented to additional follow-up. Participants were enrolled within days of HIV diagnosis and randomised within these strata to receive ART either immediately or after 24 weeks deferral .31 Enrolment of participants in the deferred group occurred before 2016, when Peru adopted the WHO “treat all” to end AIDS recommendations.32 The ongoing longitudinal study, MERLIN, follows a subset of Sabes participants on suppressive ART , with the aim of understanding how differential timing of ART initiation affects long-term outcomes .The MERLIN study follows a subset of participants in the Sabes study  with cryopreserved peripheral blood mononuclear cells (PBMCs) available. We stratified participants into three groups on the basis of whether they initiated ART at 30 days or less (acute group), at 31–90 days (early group), or more than 24 weeks (deferred group) after the EDDI  and ART initiation ; an intethe EDDI . PBMCs wAll participants provided written informed consent, and the study was approved by the ethics committee of the non-governmental organisation Impacta Salud y Educación, the Peruvian National Institute of Health, and the Institutional Review Boards of the Fred Hutchinson Cancer Research Center and the Centre Hospitalier de l’Université de Montréal.gag) and integrated  HIV DNA, and the 2-LTR junction (2-LTR circles assay) were quantified by real-time PCR, as described previously.34 Samples in which fewer than 50 000 cells were analysed were excluded from the analysis.To measure total and integrated HIV DNA and 2-long terminal repeat (LTR) circles, total CD4 cells were isolated from cryopreserved PBMCs by immunomagnetic negative magnetic selection . Pellets of enriched CD4 cells were digested with proteinase K; total  were measured by TILDA ex vivo, and the size of the inducible HIV reservoir  was measured by TILDA after in-vitro stimulation .29 HIV rples see .The main outcomes were to measure the size of the HIV reservoir before ART initiation and to assess the effect of the timing of ART initiation on the decay of HIV reservoir measures  over 4 years follow-up.10 ([HIV copies per 10⁶ CD4 cells] + 1). Results were represented as medians (IQR). To compare values between the acute, early, and deferred groups before ART initiation we used the Kruskal-Wallis test for continuous variables and the Freeman-Halton extension to Fisher’s exact test for categorical variables. The Wilcoxon matched-pairs signed rank test was used to compare values in the deferred group at enrolment and at 24 weeks thereafter (ART start date) for continuous variables and Fisher’s exact test for categorical variables. Correlations between continuous variables were assessed with Spearman’s rank correlation coefficient. The longitudinal decays in viral loads and each HIV reservoir measure were analysed by use of a mixed-effects model from the time of ART initiation up to 196 weeks in each of the three groups . Additional characteristics of the participants at enrolment are shown in To assess the seeding of the HIV reservoir, we measured total and integrated HIV DNA and 2-LTR circles, as well as the active and inducible reservoirs in CD4 cells from all participants before ART initiation. As expected, participants in the acute group had significantly higher viral loads than those in the early (p=0·056) and deferred groups  and integrated HIV DNA  compared with the deferred group.Similar to plasma viraemia, total and integrated HIV DNA levels fitted to a two-phase decay model , E. DespDuring the second-phase decay, total HIV DNA levels continued to decrease in the acute  and early groups , whereas total HIV DNA levels remained stable in the deferred group , early (31–90 days), and early chronic (6–9 months) infection, to establish whether the faster and sustained reduction of HIV reservoirs in individuals starting ART during the first month of infection extend to those initiating therapy after this point. To our knowledge, this is the first study of immediate versus deferred ART that evaluated the effect of the timing of ART initiation on HIV reservoirs in a longitudinal setting.Several studies have shown that initiating ART during acute HIV infection drastically limits the size of the HIV reservoir.39 Of note, there was a significant difference in the frequency of cells harbouring 2-LTR circles among the three groups before initiation of ART (p=0·0003), which is likely to reflect different levels of HIV replication.42We found that a large pool of HIV-infected cells was rapidly established during the first 30 days of HIV infection and remained stable, as indicated by similar baseline levels of HIV reservoir markers among the three groups. This observation is consistent with previous studies showing that the levels of total and integrated HIV DNA increase rapidly during the first few weeks of infection.39 Our study corroborates and extends these observations, as we observed a rapid decay in total and integrated HIV DNA, and in the frequency of cells with inducible HIV RNA during the first weeks of ART. This rapid initial decay was followed by a slower decay, probably reflecting heterogeneous populations of infected cells characterised by different clearance rates.43 Of note, we found that different markers of HIV persistence displayed different rates of decay during the first weeks of ART. The inducible reservoir measured by TILDA after stimulation showed a steeper decline than HIV DNA measures, possibly reflecting the preferential clearance of productively infected cells over those containing latent proviruses or viral genomes containing large defects. The faster rate of decay in reservoir markers in the acute group could have resulted from a stronger cytotoxic T lymphocyte pressure caused as a result of their preserved functions.44 This faster rate of clearance could also be due to the lower proportion of cytotoxic T lymphocyte escape mutations in the reservoir of these participants compared with those treated later;45 although, this factor remains controversial.46 As reported in our study, levels of integrated HIV DNA declined less rapidly than levels of total HIV DNA and 2-LTR circles,47 indicating that unintegrated HIV genomes are more labile, possibly because they are diluted by T-cell proliferation.Several studies have reported a rapid initial decay in the levels of total HIV DNA on ART initiation, followed by a slower decay after several weeks of therapy.15 and indicate that early HIV reservoir markers are less stable when ART is initiated within the first month of infection. This reduced stability could be attributed to the rapid clearance of short-lived CD4 effector-memory T cells, with accelerated intrinsic decay kinetics, that might be the preferential target for HIV during acute infection.48 We and others19 previously reported that the long-lived CD4 central memory T cells are protected when ART is initiated early relative to other subsets, whereas these cells constitute a large part of the HIV reservoir in people initiating ART during chronic infection . Since CD4 central memory T cells are not only long-lived, but also display exquisite proliferative capacities that can replenish the CD4 effector memory T-cell reservoir when stimulated,49 their infection might contribute to the slower decay in reservoir markers in people who delay ART initiation. Additional studies are warranted to establish whether the type of cells in which HIV persists could explain the differential decay rates between these different groups.Initiating ART within 30 days or less of infection induced a faster initial decay of all HIV reservoir markers compared with initiating ART at 31–90 days or after 24 weeks of infection. These results are consistent with those of other studies,10 did not observe a difference in the half-life of total HIV DNA between individuals who initiated ART before or after seroconversion . In this previous study, decay of HIV DNA was evaluated in bulk PBMCs, whereas enriched CD4 cells were used in our analyses, which suggests that the apparent differences between studies could be attributed to CD4 cell recovery after ART initiation. Additionally, differences in ART regimens between studies could affect viral decay, and thereby contribute to these discrepancies.Of note, the benefit of early ART was not limited to those initiating treatment during the first month of infection, since the first-phase decay of total and integrated HIV DNA was faster in individuals who initiated ART at 31–90 days after infection compared with those who initiated ART after 24 weeks. In contrast to our findings, Strain and colleagues29 In agreement with several studies showing a rapid decay in the size of the replication-competent reservoir, measured by the quantitative viral outgrowth assay,50 we observed a rapid initial decay in TILDA measures, which was faster in the acute group compared with the early and deferred groups. This observation contrasts that of a previous study, which used a quantitative viral outgrowth assay to measure the HIV reservoir, showing that the decay rates in infectious units per million cells were similar in acutely and chronically treated participants.12 Several factors could explain this difference. First, TILDA does not measure replication-competent HIV and is likely to overestimate the actual size of the viral reservoir. Second, we used total CD4 cells (as opposed to resting CD4 cells) in our analysis, thereby including activated CD4 cells, which can have a faster rate of decay than resting cells.We used TILDA measures to assess the transcriptionally competent and inducible reservoir as a surrogate for the quantitative viral outgrowth assay.17 suggesting that initiation of therapy during the first 6–9 months of infection might still be beneficial.Since our study did not include individuals with chronic infection, we cannot comment on the potential beneficial effects of ART initiation during early chronic infection  compared with infection of longer or unknown duration. Of note, other studies have not found a significant decrease in integrated HIV DNA levels in individuals starting ART during chronic infection,16 Nonetheless, and in accordance with previous studies,26 a measurable decay in integrated HIV DNA after long-term therapy was observed exclusively in participants initiating treatment within 30 days or less of infection. This observation indicates that acute ART initiation not only accelerates initial decay in the pool of infected cells, but also exerts long-term effects on the HIV reservoir. These effects could be attributed to the more labile nature of the infected cells in these individuals, in whom the long-lived CD4 central memory T cells are relatively protected by acute ART initiation.19 Although the mechanisms underlying the relative protection of CD4 central memory T cells during acute infection remain to be identified, one can hypothesise that the susceptibility of these cells increases after months of active HIV replication as a result of enhanced bystander T-cell activation. Alternatively, since antigen-driven proliferation contributes to the maintenance of the HIV reservoir,51 it is also possible that the reservoir in acutely treated participants is established in a small number of cells with a restricted number of antigenic specificities, whereas the repertoire of the reservoir in chronically infected participants is broader, and, therefore, more prone to undergo clonal expansions.The second-phase decay rates of all reservoir markers were slower than the first-phase decay rates and relatively similar across the three groups, suggesting that early ART primarily influences the slope of the first-phase decay.52A limitation of our study is that we only included individuals assigned male sex at birth; therefore, we cannot exclude the possibility that the observed differences in the rate of decay of the reservoir might not be the same in cisgender women, in whom the reservoir has been shown to be less transcriptionally active.In conclusion, initiating ART within 30 days or less of infection is associated with a faster rate of decay in HIV reservoir markers compared with initiating therapy at 31–90 days or more than 24 weeks after infection. This notable benefit of the timing of ART initiation on the HIV reservoir suggests that the beneficial effects of early ART initiation might be more pronounced after long-term therapy. In addition, these observations suggest that promoting frequent HIV screening followed by prompt initiation of ART, particularly in individuals at a high risk of acquiring HIV, could provide opportunities to accelerate clearance of the HIV reservoir.1


There is a rapidly increasing prevalence of obesity and related metabolic disorders such as type 2 diabetes worldwide. White adipose tissue (WAT) stores excess energy, whereas brown and beige adipose tissues consume energy to generate heat in the process of thermogenesis. Adaptive thermogenesis occurs in response to environmental cues as a means of generating heat by dissipating stored chemical energy. Due to its cumulative nature, very small differences in energy expenditure from adaptive thermogenesis can have a significant impact on systemic metabolism over time. Targeting brown adipose tissue (BAT) activation and converting WAT to beige fat as a method to increase energy expenditure is one of the promising strategies to combat obesity. In this review, we discuss the activation of the thermogenic process in response to physiological conditions. We highlight recent advances in harnessing the therapeutic potential of thermogenic adipocytes by genetic, pharmacological and cell-based approaches in the treatment of obesity and metabolic disorders in mice and the human. Obesity is defined as a chronic disease in which body fat accumulation promotes adipose tissue dysfunction and results in many negative metabolic and psychosocial health consequences . AccordiThere are two types of adipose tissues in the human body including brown adipose tissue (BAT) and white adipose tissue (WAT). BAT is characterized by having multiple, smaller lipid droplets and containing many more mitochondria in comparison to WAT. This allows brown adipocytes to burn energy via thermogenesis. The primary gene product involved in thermogenesis is uncoupling protein 1 (UCP1). UCP1 is exclusively expressed in BAT and works by uncoupling oxidative phosphorylation from mitochondrial respiration, which results in heat production . AdditioWhite adipocytes are characterized by a large unilocular lipid droplet that inhibits most of the space in the cell and possesses much fewer mitochondria. White adipocytes are responsible for storing energy within the body in the form of triglycerides and are commonly expanded in obese subjects. Two major types of WAT, which are subcutaneous and visceral white fats, can be found beneath the skin and surrounds the internal organs, respectively. Visceral white fats have been suggested to be associated with the risk of T2D and cardiovascular disease. Subcutaneous white fats are potentially able to be induced to acquire the features and thermogenic functions of BAT, which are termed as beige or brown-like adipocytes. Converting white to beige adipocytes, which is also known as the beiging or browning process, can be stimulated by cold acclimation, exercise training or pharmacologic activation of the β-adrenergic receptors . Two pos18F-fluorodeoxyglucose (18F-FDG), indicating high glucose uptake [Thermogenic adipocytes including brown and beige adipocytes are previously considered non-existent in adult humans. However, this concept was later modified when studies had demonstrated that active thermogenic adipocytes are actually present in adult humans. Using positron emission tomography (PET) and computed tomography (CT) scanning, the areas of thermogenic adipocytes show the significant presence of e uptake ,7,8. Adde uptake .Given the role of thermogenic adipocytes in energy expenditure and modulation of glucose and lipid metabolism and its crosstalk with other tissues, thermogenic adipocytes have been considered a promising therapeutic target to combat obesity and metabolic disorders. Overweight and obese subjects were found to have decreased thermogenic adipocytes activity in comparison to lean and healthy subjects ,10. SeveCold is the most potent stimulus to activate BAT function and induces the conversion of WAT to beige fat through stimulating the β adrenergic signaling in both humans and mice. However, cold exposure is inconvenient, uncomfortable and an unrealistic intervention to exert at bedside. In addition, cold may induce other unwanted effects such as high blood pressure . Therefoβ3-adrenergic receptor (β3-AR) agonists are potent activators of BAT thermogenesis and WAT browning in rodents. However, several attempts to develop β3-AR agonists for application in humans have not yet been successful. Mirabegron (Myrbetriq) is a β3-AR agonist approved by U.S. Food and Drug Administration (FDA) for the treatment of overactive bladder (OAB) by relaxing the bladder muscles and ameliorating the symptoms . Acute s18FDG-PET scan, Yoneshiro et al. showed that dietary supplementation of capsaicinoids enhanced glucose uptake in the BAT region and that activation of BAT was highly correlated with the increase in energy expenditure of the human subject [2+ ions and downstream signaling. TRPV1 deletion completely blocks the capsaicin-mediated anti-obesity effects in mice [Capsaicinoids include capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin and they are a group of phenolic alkaloids and active components in chili peppers . Interes subject . Capsaic in mice . In addi in mice . These fResveratrol  belongs to a class of polyphenolic compounds called stilbenes. RSV is naturally found in the roots of white hellebore, mulberries, red wine, grapes and peanuts. Administration of RSV was shown to induce beige adipocytes formation in WAT resulting in enhanced glucose uptake and energy consumption in vitro and in vivo . MechaniCurcumin, also called diferuloylmethane, is a yellow pigment hydrophobic polyphenol found in the extracts of Turmeric roots, which is a flowering plant of the ginger family. It is best known for its anti-inflammatory function and the ability to increase the antioxidant capacity. Recently, curcumin supplementation has revealed anti-obesity effect in animal models. Lone et al. demonstrated that the dietary curcumin induces browning phenotypes in WAT by the upregulation of the expression of brown fat-specific genes . This efIt has been established that thyroid hormones (THs) activate thermogenic functions in BAT of mice. Considering the translational implication, studies have tested whether THs or their analogs (levothyroxine and liothyronine) can be used as therapeutics in humans. It was found that treatment of levothyroxine could enhance the basal energy expenditure and glucose uptake of BAT in patients with thyroidectomy (NCT02499471) . In anot18F-FDG uptake in both fasting basal conditions and β-AR stimulation as compared with the placebo group (NCT02457897) [In light of the controversial results in glucocorticoids (GCs)-mediated BAT activity in rodents and humans, clinical trials have been performed to evaluate the therapeutic roles of GC analogs (hydrocortisone and prednisolone) on BAT function. In a clinical trial applying acute hydrocortisone for 14 h, healthy individuals displayed activation of supraclavicular BAT with higher skin temperature and roup NCT057897 [242457897) . The disGlucagon-like peptide 1 (GLP-1) was found to activate BAT thermogenesis in mice ,39, wherBone morphogenetic protein 7 (BMP7) promotes the commitment of mesenchymal progenitor cells to a brown adipocyte lineage and plays a crucial role in embryonic brown fat development in mice . In two Fibroblast growth factor 21 (FGF21) administration in mice was found to improve insulin-stimulated glucose uptake and activate thermogenesis in brown/beige adipocytes . SimilarCertain genes have been targeted to activate BAT function and promote the browning of WAT. There is no doubt that the activation of UCP1-mediated thermogenesis is an efficient method to dissipate excess energy and consume fuels to provide metabolic health benefits . Gene-baMice that ectopically expressed UCP1 in skeletal muscle ,48 or adNatural serotypes of adeno-associated virus (AAV) can be used for specific delivery to liver and muscle, but not for WAT and BAT. Recent efforts have focused on engineering the AAV vectors to create novel recombinant AAVs (rAAV) with diverse characteristics including specific targeting and low immunogenicity. Huang et al. engineered a hybrid serotype of rAAV, Rec2. After oral administration, Rec2 rAAV tended to preferentially target BAT with higher efficiency and specificity compared to the other natural serotypes of AAVs. The Rec2 rAAV successfully and specifically delivered the VEGF transgene to BAT and promoted thermogenesis. The finding suggested that engineered AAVs, such as Rec2, may provide a potent and specific delivery system to manipulate BAT for therapeutic applications .MicroRNAs (miRNAs) control gene expression by decreasing the mRNA stability or inhibition of the translation of mRNA. Recently, miRNAs-based therapy has been successfully applied to treat several diseases . The regThe CRISPR/Cas9 system presents a powerful strategy for genome editing in mammalian cells . SeveralFor the improvement of the CRISPR activation techniques, a nuclease-deactivated Cas9 (dCas9) is fused with transactivation domains and directed by a single guide RNA (sgRNA) targeting a specific promoter where this synthetic transcriptional complex can drive the expression of the endogenous gene . This apDelivery of ribonucleoprotein (RNP) for the gene therapy, especially in CRISPR/Cas9 genome editing, offers several advantages over viral delivery. Unlike the uncontrolled transgene integration and prolonged expression caused by using viral delivery, RNP complex is rapidly degraded and therefore enables transient genetic manipulation, limiting the off-target effect and reducing the immune response. Considering that the transcriptional and translational machineries in some cell types are not compatible with expressing the transgene driven by certain promoters, introducing the RNP complexes enables the immediate gene editing and skips the transcription or translation of Cas9 and sgRNA . RecentlCell-based therapies offer treatment strategies for patients with diseases that the existing pharmaceuticals cannot adequately address. One potential benefit of a cell-based approach, compared to strategies based on single molecules, is that the cells serve as a “living factory” to provide a more comprehensive and persistent therapeutic effect. Autologous cell therapy is a preferred therapeutic intervention in which cells are taken from an individual and administered into the same person to minimize the immune rejection. Autologous cell-based therapies have been an active area of research and are moving towards commercial development and patient access partially due to the recent advancements in the delivery systems and genome engineering methods such as CRISPR/Cas9 ,71. HereGiven the role of BAT as a secretory organ that communicates with other tissues to modulate systemic metabolism, the interest has grown in exploring BAT transplantation to provide therapeutic benefits in patients with metabolic disorders. In type 1 diabetes (T1D), transplantation of insulin-secreting β-cells to restore the insulin level is commonly used. In addition to the dysfunction of pancreatic cells, T1D patients also suffer from the loss of adipose tissues. Embryonic BAT transplantation in the subcutaneous space of STZ-induced T1D mice significantly improves glycemic control, normalizes glucose tolerance and reduces tissue inflammation. The effect has been proposed to be mediated by insulin-like growth factor 1 (IGF1) secretion from transplanted BAT ,73.Other studies demonstrated that the transplantation of adult BAT into the dorsal subcutaneous region of diet-induced obese  or genetIn clinical applications, the unlimited resource of cells provides stem cell therapy a more feasible strategy than tissue transplantation. Considering the limited amount of BAT and brown adipocyte progenitor cells in the adult human, the approach using white adipocyte progenitors or even less committed stem cells seem more applicable.Overexpression of PRDM16 and C/EBPβ, which are two important factors involved in thermogenic differentiation, stimulated fibroblasts to differentiate into adipocytes and forms an ectopic fat pad with brown fat-like characteristics after transplanting into mice. These differentiated brown-like adipocytes have been shown to be functional and able to uptake glucose . AlternaIt is worth noting that different transplantation studies have identified distinct secreted factors mediating the activation of endogenous tissues and the modulation of systemic metabolism. The differences can be attributed to the use of different species (mouse or human), different transplants  and different transplantation sites . Each study examined a unique condition that generated certain signals contributing to the wide range of effects and phenotypes reported.In elderly subjects or lipodystrophy patients, the number and the differentiation capacity of adipocyte progenitors and stem cells are limited . It is dThermogenic adipocytes, which include brown and beige adipocytes, arise from distinct origins during embryonic development; however, they share similar effects in thermoregulation and nutrients utilization. Secreted factors from brown/beige adipocytes regulate the function of adipocytes and other cell types could control systemic metabolism through the whole body. This review has focused on the potential of thermogenic adipocytes in the prevention and treatment of obesity, diabetes and related metabolic disorders. We have discussed the advantages and disadvantages of different approaches including genetic, pharmacological and cell-based strategies in the activation or increase in thermogenic adipocytes.Although multiple therapeutic strategies targeting brown/beige fat have been shown to work well in the rodent models, most of these tools are not applicable to humans. There is a significant knowledge gap in translational application from mouse to the human, especially considering the differences in brown fat between the two species. For example, Blondin et al.  recentlyIn order to mimic human conditions in mice, de Jong et al. recently performed studies in middle-aged mice housed in a thermoneutral condition (30 °C) and fed them with a 45% high-fat diet and concluded that human BAT is similar to murine classical brown fat in terms of cellular, molecular biological and morphological characteristics . AlthougRegarding therapeutic approaches aiming to increase the amount or activity of thermogenic adipocytes, cell-based and gene therapies also offer feasible therapeutic options. Autologous cell therapy is considered a safe and less invasive approach. Allergenic cell therapy can provide a universal cell source when the precursor cells derived from the patient is limited; however, the rejection problem should be solved. Gene therapy using the viral delivery system has been applied in many non-metabolic diseases due to its high efficacy. Importantly, unintended genome integration, high immunogenicity and safety issues associated with gene delivery need to be addressed. Other non-insertional genetic approaches, such as microRNA-based or mRNA-based medicine, which possess a low risk of permanent genomic alteration, may be more applicable in humans. Recent advances in mRNA modification and the delivery system could greatly improve the clinical application. In addition, synthetic mRNA encapsulated with biodegradable lipid nanoparticles was shown to enhance the stability and translatability of mRNA and was successfully applied to the treatment of a genetic disorder in the liver . HoweverIn the development of therapeutics using both genetic or pharmacological approaches, tissue-specifc and cell type-specific targeting strategies are greatly desired. Improving targeting specificity enables the reduction in effective dosages and off-target effects and results in cost-effectiveness and safety of the treatment. Customization of gene expression with the cell-specific or inducible promoters renders it possible for selective genetic manipulation in a spatio-temporal manner. Another strategy is to engineer the designated surface receptors or ligands on nanoparticles, carriers or Cas9 protein to be selectively delivered to certain cells via specific binding. In order to specifically target WAT, two adipose vasculature targeting peptides, which are iRGD and P3, have been conjugated to the membrane of nanoparticles. iRGD and P3 bind to integrin αvβ3/β5 receptors and prohibitin, respectively .Moreover, genetic manipulation needs to be strictly controlled in spatial and temporal manners. Recently, Chen et al. applied near-infrared optogenetics and Au nanorod in CRISPR/Cas9 system to regulate gene editing with high precision and spatial specificity. In their design, Cas9 expression is driven by a heat-inducible promoter and is encapsulated by a cationic polymer-coated Au nanorod. Au nanorod not only serves as a carrier to efficiently deliver the plasmid but also works as a photothermal transducer to produce heat for Cas9 expression after receiving the photonic energy from the near-infrared light. Fine-tuning of the optogenetic system offers the spatial and temporal controls required for CRISPR/Cas9-based gene therapy .In conclusion, by combining the current advances in the fundamental body of knowledge and new technologies, harnessing the therapeutic potential of thermogenic adipocytes for combating obesity and related metabolic disease proves to be promising.


Over the past two decades, organic molecules adsorbed on atomically defined metal surfaces have been intensively studied to obtain an in-depth understanding of their self-assembly behavior, on-surface reactivity, as well as their structural and electronic properties –6. An imThe molecule–substrate decoupling can either be achieved by suitable molecular design or manipulation of the molecular building blocks, or passivation of the substrate. Molecular engineering approaches include rigid spacers added to the molecular core –10 or sp2O3 [hBN) [2 [Ultrathin semiconducting or insulating decoupling layers can be epitaxially grown as mono- and multilayers on many metallic substrates by either physical or chemical vapor deposition. Among others, ultrathin dielectric layers of either alkali halides  or meta2O3 , and CuO2O3 ), or nit2O3 ) have beO3 [hBN) –23, grapO3 [hBN) –27, and [hBN) [2 , have em[hBN) [2 –32. Addi[hBN) [2 .For semiconductors, for example, bare silicon or germanium, electronic decoupling of molecules can be achieved by either the growth of ultrathin dielectric layers on top of the surface –35 or a Most of these ultrathin interfacial layers significantly reduce both the molecular adsorption energy and the hybridization of molecular states with the electronic bands in metals and semiconductors. However, charge transport is not completely inhibited, and electrons can still tunnel from the metal through these decoupling layers to the organic molecules and vice versa. This has the great advantage that these systems can still be examined by scanning tunneling microscopy (STM) and spectroscopy (STS), which gives insight into structural and electronic properties of individual molecules. For applications such vertical tunneling through the interfacial layers can be undesirable since it prevents the current from flowing across electrode–molecule–electrode junctions  or leads2, and calcite) have mostly served as model systems. To a lesser extent, metal oxides have also been used, for which defects and charging often pose additional challenges [For studying molecules on bulk insulators, ionic crystals  of molecular resonances observed in tunneling spectroscopy since the hybridization of molecular states with the ones of the metallic/semiconducting support is prevented. Thereby, it became feasible to investigate molecular orbitals , observe10This Thematic Issue highlights recent experimental and theoretical developments in realizing and understanding physically and electronically decoupled single molecules and molecular assemblies on surfaces. Several decoupling strategies at the solid–vacuum and solid–liquid interface were explored to elucidate structural, electronic, vibronic, and chemical properties of decoupled molecular structures.S)-camphor on Cu(111) [Physical decoupling by molecular design often relies on non-planar adsorbates with bulky spacer groups, which can adopt various conformations. From a theoretical point of view, finding the energetically most stable conformational structure can be challenging and costly because conventional atomistic simulations are often limited to the partial exploration of the potential energy landscape due to the complexity of the system. Recently developed structure search methods that combine machine learning with density functional theory provide the possibility of reliable structure identification of non-planar molecules, as demonstrated for the example of (1 Cu(111) . Such con-tetradecane) and tip material , which indicates that the ligands have to be decoupled from the substrates [With an appropriate molecular design, the built-in functionality of the active part of the molecule can be preserved upon adsorption on a surface. An example of the preservation of catalytic properties is demonstrated for the redox behavior of manganese porphyrins at the solid–liquid interface. Redox reactions at the axial ligands attached to the metal center of the porphyrin were observed regardless of the type of surface (highly oriented pyrolytic graphite (HOPG) and Au surfaces were used), solvent  as substrate that the HOMO of tetracyanoquinodimethane (TNCQ) is not decoupled because it is located in the MoS2 valence band, while the lowest unoccupied molecular orbital narrows but still suffers from lifetime broadening because it is situated at the conduction band onset of MoS2. Despite this, the vibronic states of the transiently negatively charged TCNQ could be resolved by STS.The semiconducting 2D material MoSd et al.  found ushBN is an insulator and has therefore been widely used to decouple organic molecules from metal substrates. Three articles within this Thematic Issue successfully employed hBN to investigate the pristine properties of particular molecules. Schaal et al. [hBN on Ni(111) electronically decoupled tetraphenyldibenzoperiflanthene such that the molecular vibronic progression was observable by in situ differential reflectance spectroscopy, which is otherwise only achieved for multilayers on the bare Ni. On hBN/Cu(111), Zimmermann et al. [hBN, ordered molecular films were observed. The decoupling even allowed for the formation of complex self-assemblies such as kagome lattices by tuning the number and position of the substituents of the pyrenes derivatives [l et al.  showed tn et al.  could vin et al.  measuredivatives .Metal oxide thin films on top of metal substrates are another interesting class of ultrathin interfacial layers to decouple organic molecules and to enable the study of their electronic properties without the contribution of the underlying metal substrate. Hurdax et al. reported that both charged and neutral species of sexiphenyl can co-exist on thin dielectric MgO films on Ag(100) . Due to 2 thin films on Si(111) [2, and on a thicker CaF2 layer. While isolated PTCDA molecules were pinned to defects on Si and also on the thin CaF2 layer, PTCDA was physically decoupled via the thicker CaF2 films and self-assembled into small islands. For FePc on H-passivated Ge(001), efficient physical decoupling facilitated the growth of large islands with upright oriented molecules, similar to their arrangement in molecular crystals.Physical decoupling of molecules from a semiconducting substrate is discussed for the example of both insulating CaF Si(111)  and hydr Si(111) . In the In summary, the articles collected in this Thematic Issue highlight recent experimental and theoretical developments in the atomic- and molecular-scale understanding of physically and electronically decoupled single molecules and molecular assemblies on surfaces. Over the last decade, significant progress in this field led to a manifold of decoupling strategies for single molecules and surface-supported molecular architectures. Decoupling strategies are highly relevant to preserve the intrinsic structural, electronic, and optical properties of the molecules for performing insightful fundamental surface-science-based studies on them and, thus, play a crucial role in designing new molecule-based electronic and optoelectronic devices.Sabine Maier and Meike StöhrErlangen and Groningen, July 2021


In this review article, we will first provide a brief overview of the ErbB receptor–ligand system and its importance in developmental and physiological processes. We will then review the literature regarding the role of ErbB receptors and their ligands in the maladaptive remodeling of lung tissue, with special emphasis on idiopathic pulmonary fibrosis (IPF). Here we will focus on the pathways and cellular processes contributing to epithelial–mesenchymal miscommunication seen in this pathology. We will also provide an overview of the in vivo studies addressing the efficacy of different ErbB signaling inhibitors in experimental models of lung injury and highlight how such studies may contribute to our understanding of ErbB biology in the lung. Finally, we will discuss what we learned from clinical applications of the ErbB1 signaling inhibitors in cancer in order to advance clinical trials in IPF. The epidermal growth factor (EGF) receptor (EGFR), belongs to the family of the ErbB tyrosine kinase receptors ,2. EGFR EGF is a prototype member of the ErbB ligand family. This polypeptide was originally identified in the submaxillary glands of mice and the urine of humans. EGF was discovered by Stanley Cohen while working with Rita Levi-Montalcini on nerve growth factors. For these discoveries, Stanley Cohen and Rita Levi-Montalcini were awarded the Nobel Prize in Physiology or Medicine in 1986  Figure . The NRGSo far, there is no known ligand for the ErbB2 receptor , howeverUpon ligand binding, ErbB receptors trigger phosphorylation of diverse effector proteins and activation of multiple downstream signaling pathways. There is a number of excellent reviews describing downstream signaling pathways initiated by ErbB receptors for those interested in this topic ,15. HereBesides its role in many developmental and physiological processes, overactivation of ErbB signaling has been widely described in many forms of cancer, including glioblastoma  and lungIn this review, we will focus on the ErbB signaling in lung fibrosis, with special emphasis on idiopathic pulmonary fibrosis (IPF). We will discuss the implications of the ErbB signaling in processes that are hallmarks of the maladaptive remodeling of lung tissue. Lastly, we will critically discuss recent advances and future perspectives in targeting the ErbB signaling for lung fibrosis therapy.MUC5B promoter variant rs35705950 is the dominant risk factor for disease development [Idiopathic pulmonary fibrosis (IPF) is one of the most common forms of diffuse parenchymal lung diseases and is characterized by excessive deposition of extracellular matrix proteins in the lung . IPF is elopment . Althougelopment  linked MIncreased expression of several cytokine/growth factors have been considered to drive profibrotic processes in the lung, including transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α). These mediators contribute to lung tissue scarring by deregulating activation, survival, proliferation, and differentiation of a variety of cells, including mesenchymal and epithelial cells ,55. For Despite the approval of pirfenidone  and nintTargeting ErbB receptors and their ligands may serve as a potential therapeutic option for IPF, in particular, that different elements of the ErbB signaling can be pharmacologically targeted. However, a complex ligand–receptor network and the involvement of the ErbB signaling in the tissue regenerative process may encounter some unexpected surprises, thus further studies critically evaluating the role of ErbB receptors and their ligands in adaptive versus maladaptive remodeling of lung tissue are urgently needed.ErbB receptors and their ligands are expressed in a large variety of human tissues including the epithelial cells of the lung . Under pIncreased expression of various ErbB ligands is associated with fibrosis development in multiple organs, including, lung, liver, or pancreas. For example, overexpression of HB-EGF and AREG causes pancreatic fibrosis ,68, whilBesides being overexpressed in many types of cancer, ErbB1 is also upregulated in lung epithelial cells from patients with different forms of pulmonary fibrosis . In IPF,After the introduction of ErbB1 TKI to cancer therapy, the discussion on their repurposing and usage in the treatment of other hyperproliferative diseases, including lung fibrosis, began. Quickly, first studies demonstrated that tyrphostin AG1478 reduces proliferation of LF and attenuates pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide in rats . AnotherEGFR polymorphism leading to the genetic susceptibility to the treatment with ErbB1 TKI in the Japanese was not observed [Development of acute lung injury and ILD in non-small cell lung cancer (NSCLC) patients receiving gefitinib demonstrates possible deleterious effects of the ErbB1 signaling inhibition ,79. Inteobserved . Pre-exiobserved ,84. Consobserved . Currentobserved .Among all ErbB1 ligands, TGF-α is the one with a well-described function in pulmonary fibrosis. TGF-α was found to be overexpressed in ATII cells, endothelial cells and fibroblasts in the lungs of IPF patients . In addiIn line with these observations, ErbB1 TKI, gefitinib, partially reduced collagen levels and improved lung compliance, tissue and airway elastance, and airway resistance in mice overexpressing TGF-α under tetracycline-inducible CCSP (rtTA-CCSP) promoter . These c4) [Amphiregulin, another ErbB1 ligand, was discussed in the context of maladaptive remodeling of the liver and lung. In this respect, AREG-deficient mice were found to be protected against liver fibrosis induced by chronic administration of carbon tetrachloride (CCl4) . To deci4)  reported4) . Consist4) ,97. Andr4) . IntercoMUC5B promotor variant underscores this assumption. The complexity of AREG cellular effects is underscored by the recent publication by Stancil et al., who showed the role of the AREG–ErbB1 axis in the jamming–unjamming of airway epithelial cells in IPF. Jamming transition describes a process of epithelial cell transformation from migratory (unjammed) to non-migratory (jammed) status in the absence of wounding or cell-type changes. This transition is believed to play an important role during embryogenesis, in processes such as axis elongation and tissue development ,99,100. Besides its “bad” role in lung fibrosis, AREG was also found to contribute to the restoration of tissue homeostasis after acute lung injury driven by infection. Minutti et al.  showed tvia the nonfunctional ErbB2–ErbB3 complex. These findings are corroborated by Faress et al. [Another ErbB receptor that was linked to pulmonary fibrosis is ErbB2. Besides being an important oncogene in breast and ovarian cancer , ErbB2 ws et al. , who repThe important role of ErbB2 in bronchial epithelial cell differentiation and proliferation was shown by Vermeer et al. . These aThe ErbB3–NRG1-α axis was also discussed in the context of alveolar bronchiolization seen in the lungs of IPF patients. In these patients, NRG1-α was detected in epithelial cells lining honeycombing areas, as well as in normal submucosal glands . In addiTaken together, it seems that ErbB2–ErbB3 activation is essential for the differentiation of airway epithelial cells and their integrity. Overactivation of this receptor complex system may induce abnormal behavior of airway epithelial cells thereby contributing to the honeycomb cyst formation and fibrosis progression. Because of the risks associated with the ErbB2–ErbB3 complex inhibition, close patient monitoring and patient categorization have to be taken into account when considering an anti-ErbB2–ErbB3 therapy in IPF.A growing body of evidence suggests the pivotal role of the ErbB-ligand system in irreversible lung tissue scarring . The ErbIn addition, a lesson has to be also drawn from the remarkable progress in understanding the ErbB biology in cancer. It is now clear that the results of clinical trials can only be improved by taking into account a number of important issues. First, the effects of the targeted therapy may be weakened because of differences in etiology and heterogeneity. Furthermore, stratification of the patients according to a predominant disease mechanism have to be considered. Molecular endotyping should be integrated into the protocols of clinical trials. This strategy may promote the prudent use of novel targeted therapies. Finally, identification of the factors that can predict drug response or resistance will play a fundamental role to tailor individual ErbB-based therapy regimens.


Electronic medical records (EMRs) are integrated information sources generated by health care professionals (HCPs) from various health care information systems. EMRs play crucial roles in improving the quality of care and medical decision-making and in facilitating cross-hospital health information exchange. Although many hospitals have invested considerable resources and efforts to develop EMRs for several years, the factors affecting the long-term success of EMRs, particularly in the EMR infusion stage, remain unclear.The aim of this study was to investigate the effects of technology, user, and task characteristics on EMR infusion to determine the factors that largely affect EMR infusion. In addition, we examined the effect of EMR infusion on individual HCP performance.A questionnaire survey was used to collect data from HCPs with >6 months experience in using EMRs in a Taiwanese teaching hospital. A total of 316 questionnaires were distributed and 211 complete copies were returned, yielding a valid response rate of 66.8%. The collected data were further analyzed using WarpPLS 5.0.2R=0.771) was mainly affected by user habits (β=.411), portability (β=.217), personal innovativeness (β=.198), technostress (β=.169), and time criticality (β=.168), and individual performance (2R=0.541) was affected by EMR infusion (β=.735). This finding indicated that user , technology (portability), and task  characteristics have major effects on EMR infusion. Furthermore, the results indicated that EMR infusion positively affects individual performance.EMR infusion (The factors identified in this study can extend information systems infusion theory and provide useful insights for the further improvement of EMR development in hospitals and by the government, specifically in its infusion stage. In addition, the developed instrument can be used as an assessment tool to identify the key factors for EMR infusion, and to evaluate the extent of EMR infusion and the individual performance of hospitals that have implemented EMR systems. Moreover, the results can help governments to understand the urgent needs of hospitals in implementing EMR systems, provide sufficient resources and support to improve the incentives of EMR development, and develop adequate EMR policies for the meaningful use of electronic health records among hospitals and clinics. Electronic medical records (EMRs), as an important health information technology (HIT), have been developed to solve problems arising from the use of paper medical records, including the difficulty in searching for information, incompleteness of information, illegibility of handwriting, difficulty in management and storage, and inaccessibility . EMRs arPrior studies found positive correlations between information technology (IT)/information system (IS) utilization and individual and organizational performance -12. WithAlthough EMRs can provide clinical and operational benefits, EMR adoption is lagging because of user resistance and other barriers . Most EMNumbeo  reportedMoreover, the initial success of EMR adoption and acceptance does not ensure its long-term success in terms of its incorporation into the daily operating procedures of hospitals in the technology infusion stage. Therefore, the purpose of this study was to understand the determinants and consequences (performance impacts) of EMR infusion at the individual level. The research questions addressed in this study were: (1) What are the salient factors (motivations) of technology, task, and user influencing EMR infusion by HCPs? (2) How do these factors (motivations) influence EMR infusion and performance?ITs and ISs have the potential to substantially improve the operational effectiveness and efficiency of an organization if used appropriately. IT implementation is a dynamic IT adaption process in organizations with various stages, including initiation, adoption, adaptation, acceptance, routinization, and infusion, as identified by Cooper and Zmud  based onSaga and Zmud  investigSome well-known theoretical models have been proposed and validated for IT/IS postadoption stages, including technology adoption ,39,40, INg and Kim  investigPerformance evaluation is a key surrogate measure for IS success within the IS discipline in the postimplementation stage -12. GoodThe TTF model has been considered as the core of investigating IS infusion and its effect on individual performance -23. In aHIMSS Analytics defined an EMR as “an application environment composed of the clinical data repository, clinical decision support, controlled medical vocabulary, order entry, computerized provider order entry, pharmacy, and clinical documentation applications.” Moreover, the environment contains patients’ EMRs across inpatient and outpatient services, and is used by HCPs to document, monitor, and manage health care delivery within a health care organization . Boonstroptimizing the documentation of patient encounters, improving the communication of information among physicians, improving access to patient medical information, reducing errors, optimizing billing, improving reimbursement for services, forming a data repository for research and quality improvement, and reducing the use of papers.Furthermore, EMRs designed to document patient care information and enable data sharing among clinicians ,51 can dEMR systems are viewed as both EHR systems and clinical information systems in hospital settings to provide clinical, communicational, and administrative functionalities . Among tAlthough the appropriate use of EMRs can improve quality, continuity, safety, and efficiency in health care, they have not been widely adopted in health care institutions as expected because of resistance among HCPs . To undeHowever, previous studies have argued that the scope of these theories is limited because they do not address the causes related to the adoption process, specifically for the postimplementation stage ,57,60. IBhattacherjee  indicateThe aforementioned findings provide the theoretical basis for this study in terms of understanding the effects of the antecedents of EMR infusion  on the exploratory, integrative, and future (emergent) use of EMRs, and the subsequent effects on individual performance.On the basis of the results reported by Hsaio and Chen  and O’CoThe technology characteristics examined in this study included accessibility (availability), maturity, and portability; these factors determine the functionality or usability of an EMR. The task characteristics examined in this study included time criticality, interdependence, and mobility. The user characteristics examined in this study included personal innovativeness in IT, technostress, and habit. These characteristics represent individual traits and perceptions toward the use of IT.EMR infusion is the extent of the exploratory, integrative, and future use of the EMR. Individual performance is defined as the improvement in HCPs’ efficiency, effectiveness, and quality of medical activities through EMR continuance (use). O’Connor et al  and TennH1: The technology characteristics of health care professionals (HCPs) significantly affect electronic medical record (EMR) infusionH1a: The accessibility of an EMR significantly affects EMR infusionH1b: The maturity of an EMR significantly affects EMR infusionH1c: The portability of an EMR significantly affects EMR infusionH2: The task characteristics of HCPs significantly affect EMR infusionH2a: Task time criticality significantly affects EMR infusionH2b: Task interdependence significantly affects EMR infusionH2c: Task mobility significantly affects EMR infusionH3: The user characteristics of HCPs significantly affect EMR infusionH3a: Personal innovativeness in information technology significantly affects EMR infusionH3b: Technostress significantly affects EMR infusionH3c: Individual habit significantly affects EMR infusionH4: EMR infusion significantly affects individual performanceThe respondents were HCPs (doctors and nurses) who worked at different departments in the case hospital, and we elaborated rigorous instrument design processes to minimize the potential risks of common method bias derived from a single-respondent questionnaire-based survey as reported by Podsakoff et al . In the The second stage of the questionnaire design focused on the evaluation and selection of the measurement items. To evaluate the content validity of the questionnaire, the content validity index (CVI) was calculated, and a threshold value of >0.80 was recommended . Two expTo ensure that the data collected were in line with the research objectives, only HCPs with >6 months experience using the EMR in the case hospital were included as participants in this study. A total of 120 doctors and 500 nurses from hospitals in southern Taiwan were recruited. The case hospital has been using EMRs since 2009 to improve their quality of care, continuity, safety, efficiency, and medical decision-making. In addition, the case hospital obtained the EMR accreditation issued by the Ministry of Health and Welfare of Taiwan. This certification indicates that the core EMR functionality can provide the services of cross-hospital health information exchange and EHRs with the support of the existing IT and IS infrastructure.2R (ARS), average adjusted 2R (AARS), average block variance inflation factor (AVIF), average full collinearity variance inflation factor (AFVIF), Tenenhaus goodness of fit (GoF), and 2R contribution ratio (RSCR) [2R coefficients when predictors cannot adequately improve the explanatory value of each latent variable [2R effects [The collected data were further analyzed using WarpPLS 5.0 , with tho (RSCR) . Kock [7o (RSCR)  indicatevariable . Both AVvariable  mentione effects .β) and 2R value. Path coefficients represent the strength and direction of the relationship between variables, and they are meaningful to research if they achieve a statistically significant level. 2R values indicate the total variance of dependent variables explained by influencing variables, demonstrating the predictive power of the investigated model.WarpPLS 5.0 with the bootstrap resampling method was used to analyze the structural model, which was mainly evaluated using the path coefficient  of St. Martin De Porres Hospital in Taiwan before the distribution and collection of surveys. After receiving approval from the IRB of the target hospital (IRB-15B-021), we obtained voluntary and verbal consent from the study participants. The requirement to document consent was waived. The responses of HCPs were anonymous and unidentified.The questionnaire was administered to collect data from HCPs who worked at the case hospital at the time of the survey and had at least 6 months of experience in using EMRs. A total of 316 questionnaires were distributed and 211 complete copies were returned, resulting in a valid response rate of 66.8%. Voluntary participation might explain the relatively high response rate. According to the collected demographic data , most ofAs shown in We further evaluated the reliability and validity  of the instrument as suggested by previous studies ,81,82. HAs shown in 2R=0.771) was mainly affected by user habits (β=.411), portability (β=.217), personal innovativeness (β=.198), technostress (β=.169), and time criticality (β=.168), whereas individual performance (2R=0.541) was affected by EMR infusion (β=.735). This finding indicated that user , technology (portability), and task  characteristics have major influences on EMR infusion. Furthermore, the results indicated that EMR infusion positively affected individual performance. Consistent with the findings of previous IS infusion and TTF studies [β=.217) affected EMR infusion; however, the effects of accessibility and maturity on EMR infusion were not as expected. Portability is the degree of ease associated with transporting EMRs [The results revealed that EMR infusion  and mobility (β=.143) affected EMR infusion. The tasks of HCPs are complex and time-critical, and require mobility, specifically for providing services in inpatient and emergency departments. Real-time and accurate information obtained from an EMR is critical to increase efficiency and effectiveness in patient care duties [O’Connor et al  reportede duties . These fe duties ,50, inteβ=.198), technostress (β=.217), and habit (β=.411), significantly affect EMR infusion. All three user characteristics exerted significantly stronger effects compared with those of technology and task characteristics on EMR infusion. This finding implied that user characteristics are the key antecedents of EMR infusion. The individual habit of EMR use showed a consistent result with the findings of O’Connor et al [This study found that user characteristics, namely personal innovativeness . Previous studies have indicated that IT assimilation and infusion are two types of use that are beyond routine use, and refer to a deeper level of usage that enhances work tasks and performance ,23. ConsWe performed an additional analysis to determine HCPs’ performance based on EMR use. As shown in Previous studies have suggested investigating the IS infusion chain, including the antecedents of IS infusion and the outcomes of IS infusion at the individual level with broader considerations, to examine the extent to which the full potential of ITs and ISs has been embedded in an organization’s or individual’s work system ,23. ThisThis is one of the few studies to specifically focus on EMR infusion by considering technology, task, and user aspects, and to examine EMR infusion effects on individual performance. The results of this study can be helpful for extending IS infusion research and identifying critical factors affecting EMR assimilation and infusion where EMRs have been deeply incorporated into the daily operating procedures of hospitals. In addition, EMR design and implementation should meet HCP task needs, particularly for time critically and mobility, and technology needs, specifically for EMR portability. Moreover, future studies can extend the results of this study by incorporating different behavioral theories and factors as the antecedents of IS infusion, and investigating their effects on IS infusion and individual performance. We found that accessibility (availability) and maturity among technology characteristics and interdependence among task characteristics were insignificant factors for EMR infusion. Therefore, additional studies should be performed to validate the factors investigated in the research model because their effects on IS infusion may vary depending on technology, task, and user groups. In addition, inspired by Kim et al  on what The key factors identified in this study provide useful insights for the further improvement of EMR development in hospitals and the government, specifically for the infusion stage. In addition, the developed instrument can be used as an assessment tool for identifying the key considerations of EMR infusion, and for evaluating the extent of the EMR infusion and individual performance of hospitals that have implemented EMR systems. The results can help the government to understand the urgent needs of hospitals in implementing EMRs, provide sufficient resources and support for improving the incentives of EMR development, and develop adequate EMR policies for the widespread use of health information exchanges and EHRs. Future studies should focus attention on these characteristics, specifically user characteristics  and task characteristics , to further facilitate EMR infusion. Our findings indicate that the routine use of EMRs by HCPs in their daily workflow processes can reduce their technostress related to the use of EMRs and increase their perceived personal innovativeness, thus promoting EMR infusion.This study has several limitations. First, we collected samples from a regional teaching hospital in Taiwan, restricting the generalization of the findings to other medical institutions. Second, the data were derived from questionnaires provided to participants with more than 6 months experience in using EMRs. Respondents answered questions based on their perceptions, experiences, and understanding. Thus, the data collected may not be adequately objective. However, due to the nature of this study (exploratory research), the quality of the collected data is acceptable. Furthermore, this study was based on a sample of voluntary participants. This type of recruitment is not considered to have negatively affected the results because this approach is commonly used in the field.


In response to aging and disability stakeholder advocacy in California, the state’s 2018-19 budget included support for the development of a study of Californians with needs for long-term services and supports (LTSS). Existing data on LTSS most typically represents those who already use specific programs or services. Yet many programs do not uniformly collect and report data, or have little capacity to share data across different delivery systems. In response to these gaps, we developed a 15-minute follow-on survey to the 2019-2020 California Health Interview Survey (CHIS), gathering statewide population-level data to assess LTSS needs and use by Californians 18 years of age and older. This paper reports on preliminary findings from the 2019 CHIS-LTSS survey conducted with a sample of 1097 respondents. Screening questions identified respondents reporting difficulties with concentrating, remembering, or making decisions (60%), performing basic daily activities such as dressing or bathing (26%), or getting out of the house to shop or to see the doctor (52%). Nearly half of respondents (45%) reported needing help with routine care needs while 16% needed help with personal care needs. Additional findings illustrate specific LTSS needs, service use, consequences of unmet needs, financial concerns, and consumer experiences. At a time when California policy makers, program planners, and advocates are engaged in implementing a 10-year Master Plan for Aging, these findings can be used to identify and address gaps in the types of services and supports that are essential to meet the LTSS needs of older adults and people with disabilities.


Cardio- and Aphthovirus genera over 50 years ago, but the molecular basis of their function is still unknown. Truncation or complete deletion of the polyC tracts in picornaviruses compromises virulence and pathogenicity but do not affect replicative fitness in vitro, suggesting a role as “viral security” RNA element. The evidence available suggests that the presence of a long polyC tract is required for replication in immune cells, which impacts viral distribution and targeting, and, consequently, pathogenic progression. Viral attenuation achieved by reduction of the polyC tract length has been successfully used for vaccine strategies. Further elucidation of the role of the polyC tract in viral replication cycle and its connection with replication in immune cells has the potential to expand the arsenal of tools in the fight against cancer in oncolytic virotherapy (OV). Here, we review the published data on the biological significance and mechanisms of action of the polyC tract in viral pathogenesis in Cardio- and Aphthoviruses.Long polycytidine (polyC) tracts varying in length from 50 to 400 nucleotides were first described in the 5′-noncoding region (NCR) of genomes of picornaviruses belonging to the  The best-known genera are Enterovirus , Cardiovirus , Aphthovirus (foot-and-mouth disease), and Hepatovirus (hepatitis A virus). Picornaviruses are non-enveloped viruses of 30 nm in diameter, consisting of an icosahedral capsid containing a tightly packaged non-segmented, single-stranded positive-sense RNA genome that ranges from 7 to 9 kb. The overall organization of the genome is highly conserved across genera: 5′ and 3′ noncoding regions (NCRs) flanking one coding sequence that is translated directly as one polyprotein. The 5′-NCRs range between 400 and 1,500 nucleotides in length, and they are linked at their 5′ terminus to the viral protein VPg, which acts as a primer during viral RNA synthesis. The 3′-NCR is much shorter, usually in the range of 100 to 300 nucleotides, and terminates with an encoded polyadenosine tract [Fig 1).Picornaviruses comprise a large family of small RNA viruses responsible for a variety of important human and animal diseases. According to the International Committee on Taxonomy of Viruses (ICTV), picornaviruses currently consist of 147 species grouped into 63 genera, and new yet unassigned viruses are continuously identified  and type II IRES (Cardioviruses and Aphthoviruses). Despite the differences, they all consist of 5 discrete stem-loop structures named modules or domains, which contain binding sites for canonical translation factors and IRES trans-acting factors (ITAFs) required for the initiation of viral genome translation [Enteroviruses: Their 5′-NCR harbors a 3-branched cloverleaf structure that guides the assembly of the replication complex that brings together the opposite 5′ and 3′-termini of the genome—a process necessary to shut down translation and initiate negative strand synthesis [cis-acting replication element or cre: This stem-loop structure contains the conserved AAACA motif acting as a template for the addition of uridine residues to viral VPg, which is used by the viral RNA-dependent RNA polymerase as a primer for both negative- and positive-strand synthesis [cre element is frequently found in the coding region of picornaviruses [Aichivirus) has been identified as the stem-loop A of the 5′-NCR [Picornavirus genomes also show a remarkable level of functional plasticity at the RNA level. Viral genomes fold into a variety of secondary structures, giving rise to stem-loops, hairpins, clover-like structures, and pseudoknots that can combine or interact with each other to form higher level tertiary structures. The vast majority of the RNA structures identified to date are located in the NCRs at the 5′ and 3′-termini, but important RNA elements are continuously being discovered throughout the viral genomes . These Rnslation . Replicaynthesis –11. Anotynthesis . The creaviruses ,14 with aviruses . Finallye 5′-NCR .Enteroviruses responsible for causing severe diseases in human. Vaccination efforts against poliovirus using the live-attenuated Sabin 3 strain led to the discovery that a single point mutation at position 472 on domain V of the type I IRES is a major determinant of poliovirus neurovirulence and can quickly revert to wild type upon replication in the human gut, as shown by cases of vaccine-associated poliomyelitis [Enterovirus genus, Coxsackievirus group B (CVB) is known to cause acute myocarditis in human, which can be fatal. This disease can sometimes lead to a chronic inflammation of the cardiac tissue associated with the presence of CVB2 and CVB3 genomes carrying small 5′-terminal RNA deletions of approximately 10 to 50 nucleotides [pro [Other RNA motifs and structures are not directly involved in the essential steps of the viral life cycle but play important roles in the pathogenicity of the virus, shaping the symptoms, immunogenicity, persistence, biodistribution, and, eventually, the disease outcome of picornaviruses . Similarmyelitis . Anotherleotides . These ddes [pro ,21. The des [pro . Full-ledes [pro ,24.Cardioviruses and Aphthoviruses genera. More specifically, the best-known bearers of this peculiar RNA element are the FMDV in the Aphthovirus genus, EMCV, and its sub-strain Mengovirus for the Cardiovirus genus. These polycytidine (polyC) tracts have drawn attention since their discovery because the evolutionary burden of maintaining such a long repetitive noncoding sequence intuitively implies a vital function. Extensive studies have shown that the polyC is most likely another example of such “viral security” RNA elements: Deletion or truncation of the tract does not affect the viability of the viruses but greatly diminishes their virulence. The role as pathogenicity determinant, the genetic stability, and the impact on viral viability of the polyC tract differ greatly from virus to virus, which makes it even more challenging to draw conclusions on the exact function and molecular mechanism of attenuation.Long stretches of 50 to 400 cytidine residues in a row, sporadically interrupted by species-specific uridine discontinuities, have been identified in selected species of picornaviruses, all belonging to the Cardiovirus and Aphthovirus genera, namely several strains of FMDV and a sub-strain of EMCV called Mengovirus [Aphthovirus genus, equine rhinitis A virus (ERAV) and bovine rhinitis B virus (BRV-2) were reported to have polyC tracts [Aphthovirus to Erbovirus genus, also carries a polyC tract [The presence of a discrete polyC tract within the genomic RNA of picornaviruses was first reported in EMCV as a tract of about 100 nucleotides in length .This obsngovirus . More reC tracts ,28. EquiyC tract .These long stretches of polyC repeats all share the same location within the genome of the picornavirus in which they have been described, within the first 500 nucleotides of the 5′-NCR. More specifically, the polyC is localized after the first 150 nucleotides for EMCV and Mengovirus ,31, afteCardioviruses have shorter polyC tracts, with EMCV varying from 70 to 250 nucleotides and Mengovirus isolates ranging from 50 to 100 nucleotides [Cardioviruses: CnUC10 for Mengovirus and CnUCUC3UC10 for EMCV (Table 1). Accurate sequencing data for the much longer polyC tracts in Aphthoviruses strains is not available to date; therefore, the length of the polyC tract in natural strains of these viruses has only been estimated through RNAse digestions and is reported as an uninterrupted polypyrimidine segment. Consequently, it is not known whether these discontinuities are a peculiarity of Cardiovirus genus (Table 1). Similarly, the complete sequence of the polyC tract and the entire sequencing upstream of it have not been determined in ERAV, BRV-2, and ERBV; therefore, the total length of the polyC is unknown, but runs of at least 13 to 17 cytidine residues have been reported for all of them [Variation in the length of this tract among isolates and among subclones of lab strains is considerably high. FMDV polyC tract ranges from 150 to 250 nucleotides in natural isolates and shows even more variability in lab and vaccine strains ,34–37. Cleotides ,35,38. I of them –29.While other “viral security elements” in picornavirus genomes consist of highly structured RNA, polyC tracts in FMDV and EMCV are largely unstructured: Biochemical analysis showed that the polyC tract is mostly looped out and exposed, therefore not forming any secondary structure or pairing with surrounding regions ,40.Rhinovirus and Enterovirus suggests that the polyC fragment is a distinctive feature of Cardioviruses and Aphthoviruses. Interestingly, the general landscape of RNA structures and motives making up the 5′-NCRs of these viruses is extremely similar and sets them aside from other picornaviruses. In both genera, the very 5′-end of the NCR folds into a long hairpin structure known as the S-fragment, while the region immediately preceding the polyprotein is organized into IRES type II. The space in between these 2 RNA elements contains the polyC tract, flanked on either side by a series of pseudoknots . The fact that this specific combination of structures is conserved in picornaviruses carrying the polyC tract might suggest that this RNA element needs to act in concert with other RNA elements to perform its function.The absence of this tract in other genera of the picornavirus family such as First attempts at cloning full-length genomes for EMCV and Mengovirus led to the serendipitous discovery that infectious clones carrying substantial or even total deletions of the polyC tract generate viable viruses that replicate with the same kinetics and titers of the wild type in vitro, although forming slightly smaller plaques ,41–44. TTable 1). This variability is expected in RNA viruses, which notoriously have a high mutational and recombination rate [Cardio- and Aphthoviruses in the wild and suggests the presence of a mechanism to preserve the length of the polyC in an optimal range.PolyC tracts lengths vary in a virus specific manner: around 50 nucleotides for Mengovirus, 100 to 150 for EMCV, and 100 to 400 for FMDV  is viable in BHK cells and has proven to be virulent in a nonhost species, but it is not genetically stable as it evolved deletion mutants directly downstream of the 2 cytidines [Studies on FMDV support these assumptions on polyC genetic stability. Recombinant FMDV with a polyC of only 2 cytidines  and the deleted MC0 (no polyC) variants of Mengovirus, similarly to their EMCV-R counterparts C4, C9 and C20, do not show evidence of polyC length reversion even after extensive passaging in vitro [24 (C13UC10) in mice brains similarly failed to restore virulence, as none of the passages induced symptoms of Mengovirus infections in mice [24 administered as oncolytic therapy to tumor-bearing mice showed that short (C13UC10) polyC tracts can remain stable up to 4 days postinfection [24 had also been restricted by the addition of microRNA (miRNA)-targeting elements, recognized by cognate miRNA overexpressed in cardiac and neuronal tissues to further limit toxicity. Such restriction of viral replication might influence the virus’ ability to amplify the polyC tract and escape [PolyC tract truncations and deletions in s clones ,42. Trunin vitro ,42. It s in mice . However in mice . Sequencnfection . Howeverd escape . The useTable 1) [Attempts at establishing a connection between pathogenicity of natural isolates and length of the polyC tracts are intrinsically hindered by the difficulty of correctly assessing the length of the polyC. Reports of pathogenic Mengovirus, EMCV, and FMDV natural isolates carrying short polyC tracts are biased by the methodology used to sequence the tract: Reverse transcriptase and other template-dependent DNA polymerases have difficulty reproducing repetitive sequences with fidelity and inevitably shorten the tract, resulting in cDNA fragments used for sequencing that do not represent faithfully the length of the polyC tract present in the natural isolates. This is reflected by the fact that every time EMCV polyC tract sequencing has been attempted using cDNA intermediates, it resulted in a short polyC tract ranging from 8 to less than 40 nucleotides, while direct assessment of polyC tract length as it appears in the viral RNA through RNase digestion and radiolabeling always results in long tracts ranging from 110 to 160 nucleotides (Table 1) –56. ConsGiven this technical difficulty, it is not surprising that a clear relationship between polyC tract length and pathogenicity has only been established for Mengovirus, which carries the shortest—and, therefore, more easily manipulated—polyC tract.115UCUC3UC10) using constructs carrying polyC tracts of 4, 9, or 20 nucleotides shows that even important truncations of the polyC tract only cause mild attenuation of EMCV in mice [10UCUC3UC10) was 100 times more attenuated in mice than its wild type counterpart [9) showed a 200-fold attenuation [141UCUC3UC10—showed the same levels of pathogenicity, virulence, and biodistribution of their parent strain in NMRI mice [Comparisons between natural long-tract isolates and recombinant short-tract EMCVs show contradicting results, making it hard to establish a linear relationship between polyC length and pathogenicity. A thorough analysis of the role of the polyC tract length in EMCV-R strain  or a truncated polyC C13UC10 (MC24) are dramatically attenuated in mice in terms of lethal outcome, disease severity, and neuropathogenic properties [The polyC tract is unequivocally a major determinant of viral pathogenicity for Mengovirus. Mengovirus is neurotropic and induces a rapid and lethal meningoencephalomyelitis after intraperitoneal or intracerebral injection, with onset of severe neurological symptoms within the first week postinoculation and death by day 10 in mice . The M i44UC10 . Mengovioperties ,66–68. Toperties ,66–68. Toperties ,67. The operties ,70.24 has drastically reduced viral replication in the brain and induces only mild neuronal lesions, while the polyC-deleted Mengovirus MC0 reaches even lower titers in the brain and causes no detectable histopathological changes [Neuropathological properties of Mengovirus inversely correlate with the polyC tract length. Direct intracranial administration has shown that wild-type Mengovirus replicates extensively in the brain until death of the animal, causing acute encephalitis as early as 1 day postinfection, with macrophage, lymphocyte and leukocyte infiltration, and necrosis of neuronal cells ,66. Shor changes ,66.Effects on viral replication in the brain are paralleled by similar effects on viremia. Infection with wild-type Mengovirus causes high and sustained viremia in mice until death . IntrapeIn spite of poor replication rates, short-tract Mengovirus with total or partial deletions of the polyC tract are excellent immunogens: Sublethal doses of the virus lead to development of neutralizing antibodies in mice, baboons, macaques, pigs, and a variety of other mammals ,66,69,70−/− mice, which have negligible CD4+ T cells and are incapable of producing neutralizing antibodies, even though more susceptible to MC24 infection can still survive and mount a protective CD8+ T cell–mediated immune response against subsequent rechallenges with EMCV and Mengovirus, even though this protection is short lived and starts to wane after 90 days postinoculation [In addition to invoking a potent humoral immune response, short-tract Mengovirus infection elicits a cell-mediated immune response. Passive transfer of serum from immunized to naive mice is protective against challenge with EMCV but only if the neutralizing antibody titer is above a certain threshold . On the culation .Cardioviruses, is extremely sensitive to the antiviral response generated by type I IFN pathway activation [Variations in the length of the polyC tract also seem to affect the innate immune response to Mengovirus. Mengovirus, as all tivation . Infectitivation ,68. The tivation . MoreoveTogether, these preliminary findings suggest the interesting hypothesis that a long polyC tract might be a cell type–specific requirement to establish a successful infection of host immune cells, either to directly squash the antiviral immune response or to hijack immune cells to facilitate and spread the infection. What makes this idea appealing is that there is considerable evidence supporting the role of immune cells, especially monocytes and macrophages, as vessels for viral replication, persistence, and dissemination in viral pathogenesis . In caseCollectively, these data suggest that the polyC-mediated impact on virulence is heavily dependent on a host component capable of detecting this small genomic difference early during infection. The unaltered replication kinetics in vitro, modest replication in the brain and low viremia suggest that short-tract viruses are viable and capable of replicating similar to their wild type counterparts. However, at each infectious cycle, they lose more ground against the cumulative effects of innate and adaptive immune responses until the host immune system eventually wins the arms race and clears the infection. Full-length polyC tracts somehow enhance the viral pathogenic effect and are capable of overpowering the host immune system.24, results in an extremely attenuated virus, which behaves in vivo just like its Mengovirus counterpart, whereas simply reducing the number of cytidine residues to C20 in EMCV has very little effect [Fig 1). However, the evolutionary burden of maintaining such a long repetitive polyC tract in the face of restrictive genomic space constrains implies the existence of a fitness payoff. The function and the molecular mechanism at the basis of the polyC tract still elude us, but some speculations can be made .These findings highlight an important biological difference between Mengovirus on one side and the EMCV and FMDV on the other, which is especially surprising when we consider that Mengovirus is a sub-strain of EMCV. Such a difference might be explained by the fact that Mengovirus has a much shorter polyC tract, and, therefore, the biological effects of length variations are immediately apparent, while EMCV and FMDV long polyC tracts are capable of buffering much more loss in sequence. Another possible explanation is that EMCV and FMDV might have evolved redundant systems to perform the same function that in Mengovirus is associated with the polyC alone, and, therefore, are more readily capable of overcoming variations in length. An interesting observation comes from studies on chimeric Mengo-EMC viruses: Replacing the first 411 nucleotides of the 5′-NCR containing the polyC tract of EMCV with the equivalent sequence of the attenuated Mengovirus, MCe effect . This sua and b of the stem-loop IV in type I IRESs in poliovirus, Coxsackievirus B3, and rhinovirus [The first scenario sees the polyC tract as a binding site for cellular or viral factors involved in replication, translation, or packaging. Constructs comprised of type II IRESs derived from polyC-containing picornaviruses are routinely used in monocistronic and bicistronic vectors in the absence of the polyC-tract; therefore, evidence that the polyC tract directly recruits factors indispensable for translation is currently lacking. However, comparative studies conducted in both cell-free systems and cell lines demonstrated that in EMCV- and FMDV-derived vectors, type II IRES–mediated translation efficiency is heavily affected by reaction parameters as well as the species and tissue of origin of the cell line used ,83. Furtinovirus –95. PCBPinovirus ,96.b in the cloverleaf (ACCCA) and in a spacer sequence immediately downstream of the cloverleaf (U/ACCC(CC)UCCCCCA) of Enteroviruses [d of the cloverleaf and to PCBP2, forming a ternary complex that bridges the gap with the polyA-binding protein (PABP) associated with the polyA on the 3′-terminal [Cardioviruses and Aphthoviruses do not possess a cloverleaf structure, which poses the question of how do these viruses accomplish circularization. In FMDV, PCBP2 promotes viral replication [Cardioviruses and Aphthoviruses playing the same role the cloverleaf plays in Enteroviruses by recruiting the circularization complex at the 5′-terminus, with the polyC tract working as C-rich spacer region immediately downstream for the stabilization of PCBP2 binding. Further validation of this hypothesis comes from the fact that mutations in the spacer region of poliovirus also abolish neurovirulence and attenuate the virus [Even more interestingly, PCBP2 also binds to C-rich motifs on bulge oviruses ,97,98. Tterminal . The cirlication . Moreovelication . With thhe virus .Flavivirus hepatitis C virus (HCV) NCRs have been shown to be specifically recognized by cytosolic RNA sensor RIG-I [Cardioviruses and Aphthoviruses [Alphavirus 5′-NCRs capable of avoiding immune restriction by antiviral factor IFIT1 through alterations of its binding and function, even though the molecular mechanism has not been elucidated yet [Another hypothesis is that the PolyC tract could have evolved to counteract the type I IFN antiviral response. PolyU/UC sequence signatures on the or RIG-I . Even thoviruses ,74,104. ated yet . Given tCardioviruses and Aphthoviruses face, in which the polyC could provide further stabilization to the 3′-NCR/S-fragment interaction.The function of the polyC tract might be not to act as a binding motif for protein factors but to act as a structural element that allows the RNA genome to assume more favorable spatial conformations. Although additional structural studies are needed, polyC tracts are not currently known to fold into any secondary structures and are thought to loop out of an otherwise highly organized 5′-NCR ,40. ThisFinally, it is also possible that the polyC sequence per se is not the molecular motif triggering the mechanism of attenuation: The complementary polyguanosine (polyG) sequence present at the 3′-NCR of the negative-strand or even the double-stranded RNA stretches of polyC/G that come into existence temporarily during the replicative form (RF) and the replicative intermediate (RI) might also be the culprit. The use of cytidines is clearly important because replacing it with polyU or random filler has the same effect of a total deletion . To this0 and MC24 have repeatedly shown that inoculation with short-tract Mengoviruses is protective against infection with wild-type Mengovirus and EMCV [PolyC truncations to control the virulence of EMCV-Mengovirus and FMDV have obvious practical applications for the containment of the viruses themselves, which are known pathogens for swine, cloven-hoofed, and zoological animals ,109. Vacand EMCV ,70. Thesand EMCV . This stand EMCV . Howeverand EMCV . BecauseEnteroviruses, Coxsackievirus A21  and a chimeric poliovirus-human rhinovirus (PVS-RIPO), both currently in Phase I and II clinical trials as therapeutics for a variety of tumor types, alone, or in combination with immunomodulators . One of the most important conundra in OV revolves around the necessity to balance safety and efficacy: Efforts to reduce pathogenicity in order to control replication and limit side effects for the patients and transmission risks for the population might end up compromising potency and immunogenicity [24 has proven to be able to effectively infect and debulk tumor volume in a multiple myeloma model, but the neuronal and cardiac toxicity associated with high titers needed for oncolytic therapy were detrimental to mice survival [24 NCRs (MC24-NC) significantly reduced replication in the target tissues even at extremely high titers and increased the safety profile of MC24. Oncolytic therapy with MC24-NC was able to delay tumor growth and even completely clear tumors in multiple myeloma models, but its efficacy is not consistent across the board [Modulating viral pathogenicity in replication-competent viruses is also an important requirement for OV, a fairly novel platform for cancer therapy. This approach harnesses the innate propensity of viruses to selectively infect and lyse cancer cells for therapeutic purposes . Becausegenicity . Notablygenicity ,115. Becgenicity . This apgenicity –119. Thesurvival . miRNA-the board ,120. ThrAphthovirus and Cardiovirus. The mystery of the polyC tract is actually 2-fold: what are the molecular interactions involving the polyC tract happening intracellularly that impact viral replication and how does the outcome of such molecular interactions ripple down to pathogenicity. Research on this topic has mostly been held back on one hand by the intrinsic difficulty of accurately sequencing long stretches of repetitive sequence and on the other hand by the challenge of synthesizing, cloning, and maintaining long CG tracts in bacterial plasmids. These obstacles explain why it has not been easy to investigate the polyC, especially at the time of its first description almost 50 years ago, when the best tool to assess the length of the polyC was digestion with RNases. We are now in the position to shed light on this somewhat forgotten enigma, a task that has become increasingly important. Studying virulence factors in animal pathogens has an immediate application for prevention and vaccination efforts, especially for economically relevant viruses such as FMDV and EMCV, but the benefits are not limited to animal welfare. Broadening the knowledge on pathogenicity determinants in other mammals has a huge but less direct impact on human health too: first, because it leaves us better equipped to understand and deal with the threats of zoonosis and emerging pathogens, and, second, because it gives us the tools to fine-tune the design of viral vectors for applications such as OV, where the balance between safety and efficacy is based on harnessing and giving purpose to viruses’ natural inclination for destruction.The polyC tract is a unique element of picornavirus biology whose molecular function and connection to pathogenesis has eluded explanation so far. The great variability in terms of genetic stability, impact on viral viability, and virulence hints at a mechanism that is far more complex than the simplicity of this RNA element molecular design—just a stretch of cytidine residues, might lead to believe. The idea that a few nucleotides difference in length is all that these viruses need to make a difference between life and death is deeply fascinating per se, but also has deep implication on the way we think about RNA viruses. Picornaviruses, just like all small RNA viruses, cannot rely on a plethora of protein-encoding genes to perform all the functions needed to hijack and defeat host cell defenses and have therefore evolved to maximize function and information embedded in their genomes to the point that they can achieve lethality by just increasing a polyC tract by a dozen nucleotides. Investigating the RNA elements distributed along the whole length of picornavirus genomes can increase our knowledge of what kind of functions are possible for RNA molecules in general. This kind of analysis has brought the world of molecular biology numerous tools that are now extensively used to parse out biological processes—a classic example being another fundamental picornavirus structure, the IRES, now extensively used to generate polycistronic reporter genes assays. Contrary to IRES, the polyC tract is unique to few picornavirus species belonging to diverse genera, which brings the question as to why this RNA element evolved specifically in the genomic context of