Even so, recent advances using next generation sequencing (NGS) techniques have identified a genetic link between WDTCs and ATCs, suggesting a step-wise accumulation of mutations driving the loss of differentiation for most cases. In this Commentary, recent findings from an NGS study on synchronous FTC, PDTC, and ATC tumor components from the same patient are highlighted. By using whole-genome data, clonality analyses identified a chief ancestral clone carrying mutations in TP53-associated signaling networks regulating genes involved in DNA repair, with sub-clones in each tumor component that were identified also in the less differentiated, neighboring tumor. Moreover, mutational signatures suggested a general mismatch repair (MMR) deficiency along with microsatellite instability. These findings support the chained progression model of dedifferentiation in thyroid cancer, and pinpoint a central role for defective DNA repair. Since effective treatment modalities for ATCs are urgently needed, studies regarding therapeutic agents specifically targeting defective MMR in dedifferentiated thyroid carcinoma could be pursued.Well-differentiated thyroid carcinoma  are fairly slow-growing tumors with an overall low mortality due to the efficacy of combinatory surgery and postoperative radioiodine therapy. Subsets of WDTCs may dedifferentiate into poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC), of which especially the latter has an exceptionally poor patient outcome. The underlying genetics responsible for this tumor progression is only partly understood, and is complicated by the fact that subgroups of ATCs are thought to arise  Patients are usually in the older span and often present with dramatic symptoms such as a rapidly growing neck mass, dyspnea, and/or hoarseness. The diagnosis is usually pinpointed after cytological examinations of fine needle biopsy material, in which highly pleomorphic and undifferentiated tumor cell aggregates are demonstrable. The tumor is often metastasized at original presentation, and the current treatment modalities in such cases are mostly palliative in nature, with debulking surgery and combinations of radio- and chemotherapy (cisplatin or doxorubicin), as well as tyrosine kinase inhibitors constituting the most common approaches in the clinical setting,2,4-8. Less than 5% of patients with distant metastases survive more than five years; however, the chance of cure is higher if the patient only exhibits localized disease without regional or distant involvement. Notably, long-term survival of ATC patients with disseminated disease have also been described, of which some have been treated with tyrosine kinase inhibitors, while rare cases with spontaneous remission also have been reported,10.Although rare, anaplastic thyroid carcinoma (ATC) is a highly lethal form of thyroid cancerde novo, without evidence of a pre-existing, well-differentiated thyroid carcinoma ; or (2) as dedifferentiated forms of pre-existing WDTCs through an inter-stage poorly differentiated thyroid carcinoma (PDTC) de novo theory builds on the fact that patients with ATCs do not always display clinical or histological evidence of an associated WDTC, but as always in medicine - “absence of evidence is not evidence of absence”. As ATCs are highly proliferative lesions with an infiltrative growth pattern and associated inflammation, destruction of nearby tissues could in theory mask the presence of a WDTC in certain cases - making the de novo concept less persuasive. Moreover, differentiated forms of thyroid cancers are histologically present in the majority of ATC cases reported in larger studies from multi-center institutions, suggesting a linkage between these tumor forms. Arguing in favor of the de novo hypothesis, previous cytogenetic analyses have pinpointed wide-spread aneuploidy among ATCs, while concurrently observed WDTCs were largely diploid. Although this study vaguely suggests that the tumors might have evolved from different mother clones, a tumoral evolution from WDTC to ATC via progressive genomic instability could not be excluded either.ATCs have traditionally been thought to develop along two separate paths: (1) ,14. For example, two the most common recurrent mutations seen in PDTCs and ATCs are the V600E BRAF mutation as well as hotspot RAS mutations, which are mutually exclusive in ATCs (BRAF or RAS). Those mutations are also frequently observed in PTCs (BRAF and RAS) and FTCs (RAS). The occurrence of similar driver mutations in WDTCs and dedifferentiated forms of thyroid cancer could argue in favor for the chained progression theory, but does not fully exclude the de novo hypothesis either. As additional genetic events are seen in PDTCs and ATCs compared to WDTCs, some of these could influence the tumor progression in synergy, not least TP53 and TERT - two of the most commonly mutated genes in ATC,13-16. However, data from paired WES studies of WDTCs and ATCs are now accumulating, and the majority of cases seem to exhibit a common and short phylogenetic tree trunk with identical mutational profiles adjoined by disparate mutations as a consequence of the tumors separating early in clonal evolution,18. These data strongly imply that ATCs, whenever a synchronous WDTC is visualized, do in fact arise by dedifferentiation from a pre-existing WDTC, highly important information from preventive and therapeutic angles.The advent of high-throughput next-generation sequencing (NGS) techniques such as whole-exome and whole-genome sequencing (WES/WGS) has revolutionized molecular biology, and also increased our understanding of thyroid cancer dedifferentiation tremendously. By recent NGS studies, the mutational landscapes of PDTCs and ATCs have been partly deciphered, and the results indicate that a large proportion of cases exhibit common driver mutations normally seen in WDTCs. In fact, patients with WDTC exhibit an overrepresentation of rare variants in genes responsible for DNA repair, including mismatch repair (MMR) genes such as MSH6, nucleotide excision repair genes  as well as genes involved in homologous recombination repair (HRR) -22. Moreover, MMR gene dysregulation and microsatellite instability (MSI) have been found in subsets of WDTCs, adding fuel to the hypothesis that aberrant DNA repair is an important component in thyroid tumorigenesis,24.As PDTCs and ATCs in general exhibit an overall higher mutational burden than WDTCs, it is not surprising that researchers are turning their attention to defects in DNA repair systems to explain these differences. Indeed, previous reports have highlighted the potential role of aberrant DNA repair mechanisms in thyroid cancer - not least through the known exogenic coupling between ionizing radiation, oxidative stress, and the development of thyroid neoplasia,14. MSI is not uncommon, and subsets of ATCs seem to exhibit a hypermutator phenotype with an immense increase in somatic mutations - most often in tumors with defects in MMR genes, such as MLH1, MLH3, MSH5, and MSH6. Moreover, these MMR deficient, hypermutated ATCs might display a slightly better overall prognosis than MMR competent ATCs, possibly reflecting a prognostic significance of this genetic feature in clinical practice. Overall, ATCs are generally genetically unstable compared to their WDTC counterparts with a massive overrepresentation of gross genetic aberrations, but a specific coupling between ATCs and defective HRR has not yet been established,13. As of this, the reason for the increase in gross structural abnormalities observed in ATCs compared to WDTCs is not fully established.Through NGS studies in ATC, somatic mutations in DNA repair genes has been reportedet al. recently employed WGS to analyze synchronous manifestations of primary FTC, PDTC, and ATC, as well as regional lymph node metastases of the PDTC and ATC components from a single patient. The main aim was to obtain high-resolution phylogenetic data through the interrogation of both mutations as well as copy number alterations on a genome-wide scale, as previous studies focused solely on WES, allowing for a less comprehensive copy number coverage. Moreover, as the tumors analyzed were diagnosed synchronously in the same thyroid lobe from the same patient and operation, this gave the authors a unique opportunity to study the genomic progression of each tumor component without the risk of sampling bias or unrelated patient comparisons. Herein, the overall results from this study as well as associated interpretations are highlighted from both tumor progression and DNA repair perspectives.In order to deepen the understanding of the phylogenetic relations between WDTCs and ATCs as well as to investigate the role of DNA repair in this process, Paulsson et al. successfully extracted DNA from normal thyroid tissue as well as neighboring FTC, PDTC, and ATC components, in addition to disseminated PDTC and ATC lymph node metastases. This strategy required the material to be scraped from sections derived from formalin-fixated paraffin-embedded tissues rather than fresh-frozen ones, as the primary FTC, PDTC, and ATC components were bordering each other and only properly visualized using light microscopy.In order to obtain material for a comparative analysis between the different thyroid tumor types derived from the single patient, Paulsson et al. identified a number of coding mutational events in cancer-associated genes from each tumor component, which were identified as somatic after correcting for variants also present in constitutional tissue. Some of these mutations were carried along from the FTC all the way to the metastatic ATC, potentially suggesting they provided the tumor components with a selective advantage . Additional tumor-related genes were mutated at the level of the PDTC component, also identified in the neighboring ATC and both metastatic lesions , implying that one or several of these contributed to the dedifferentiation process. Furthermore, mutations in TSC1, TSC2, JAK1, and DAXX appeared at the level of ATC, making it likely that some of these genetic events influenced the development of this undifferentiated tumor type - although only the TSC1 and DAXX mutations were carried along to the metastatic ATC component.Following successful whole-genome sequencing and bioinformatics analyses, Paulsson . By doing so, the authors were able to highlight a main, truncal clone that branched off into five major sub-clones. The mutational and copy number backbone in the main clones of the primary ATC and both metastatic samples were identified as small sub-clones present already in the well-differentiated FTC - strongly suggesting that the tumor components were genetically linked. These findings also open up possibilities to identify future markers of potentially lethal sub-clones in cases of WDTC to detect cases at risk for future dedifferentiation, which would be highly warranted from a clinical standpoint. It should be noted however, that non-functional and/or non-coding mutations were not included when assessing clonality, which was deemed impossible given the sheer data volume. The authors were thus not able to detail the clonal compositions using passenger alterations, which could have been useful in separating chained clonal distribution patterns of driver gene mutations with associated co-mutations from the de novo driver gene mutations occurring in the downstream tumor components.From a clonality perspective, a staggering five-fold increase in mutational burden was noted for the PDTC component compared to the other tumors sequenced, including the ATC. The vast majority of these PDTC mutations were found to be sub-clonal, and they were not carried on to the ATC component et al. identified a truncating, heterozygous MSH2 mutation in the FTC that was carried along to all subsequent tumors analyzed. Notably, the MSH2 gene locus displayed copy-neutral LOH but retained MSH2 protein expression in the FTC, although the downstream tumor components were negative for MSH2 expression. Thus, the authors speculate that the copy neutral LOH must have preceded the MSH2 mutation in the FTC, followed by a second genetic event in the PDTC and ATC components leading to abolished MSH2 expression - perhaps by promoter hypermethylation of the wildtype allele. All tumor components exhibited MSI, with the lowest score in the FTC, and analysis of the mutational composition showed a substantial accumulation of C>T transitions compared to A>G transitions and transversions for all tumor components - a known hallmark for deficient MMR. Several significant signatures were correlated to deficient MMR when correlating the genetic data to established COSMIC mutational signatures. By in silico gene ontology analyses of impactful coding mutations in the mother clone , Paulsson et al. identified “TP53 associated transcription of DNA repair genes” as the main mutated signaling pathway. In all, these data suggest that the mutational inactivation of MSH2 gave rise to an MMR deficient profile for all tumor components.Paulsson MSH2 mutation, a missense ATM mutation was observed in the adjacent PDTC component. ATM encodes a kinase protein that regulates signaling pathways involved in DNA repair and overall genome stability through regulation of HRR, including TP53 associated signaling networks. Interestingly, as the PDTC exhibited a hypermutator phenotype compared to all other tumor components, one cannot exclude a synergic effect of combined MSH2 and ATM mutations driving the hypermutability. Of note, ATM mutations have been found recurrently in ATCs. Even so, as both ATM and TP53 gene mutations are recurrent events also in HRR competent cancer types, there was not enough evidence to suggest that the ATC sample studied was indeed HRR deficient. Furthermore, when analyzing rare constitutional variants in cancer-associated genes by interrogating the normal thyroid sample, the authors detected a homozygous MUTYH variant (rs3219468) with a minor allele frequency of < 0.04% in the general population. Interestingly, MUTYH is involved in DNA repair, especially response to oxidative DNA damage. Whether this rare variant in any way influenced the development of multiple thyroid tumors in this patient is not known.In addition to the truncating in vitro and clinical studies focusing on various adjuvant therapies have been launched in recent years, including studies using immunotherapy , epigenetic silencing , metabolic pathway interference , as well as specific gene therapies using an adenovirus vector. Immunotherapy seems especially promising given the high frequency of PD-L1 and PD-1 positive ATC cells in clinical samples, as well as initial studies suggesting partial response or stable disease in subsets of patients after immunotherapy,32. Interestingly, in unrelated tumor types, the expression of PD-L1 and the prognosis of patients receiving immune checkpoint inhibitors have been found to be influenced by the MMR status and somatic mutations in MMR genes-36. If the same is true for ATC patients is not currently known, but given the known association between aberrant MMR and the development of this highly lethal tumor type, studies on this topic are highly warranted.Given the dismal overall prognosis and the limited treatment options for ATC patients, numerous . For this reason, numerous studies have been conducted in order to identify additional therapeutic options for cancers with an MMR deficiency. In one study, the authors selectively knocked-down the function of DNA polymerases POLB and POLG in cell lines deficient in MSH2 and MHL1 respectively, causing oxidative DNA lesions and lethal double strand breaks. Moreover, cytarabine, a cytosine nucleoside analogue, has been proposed as an alternative oxidative stress-generating compound with selectivity towards MMR deficient cell lines. Interestingly, the folic acid antagonist methotrexate might work in a similar way, as studies have shown an increased lethality for MSH2 mutated tumors via the induction of oxidative stress and extensive DNA damage. To build on this, studies depicting partial responses in ATC patients administered oral methotrexate in combinations with other cytotoxic drugs have been reported, but the MMR status of these tumors was never characterized,42. Finally, one should not overlook the potential of gene therapy, for example the usage of out-of-frame “suicide genes”. When introduced in MMR deficient cells, the gene is randomly reverted back to its in-frame version due to the hypermutability, leading to apoptosis.In colorectal cancer, there is an association between MMR-deficient tumors and an observed resistance to various chemotherapeutic drugs used, not least 5-fluorouracilRecent advances in NGS techniques have facilitated the genomic interrogation of thyroid tumors, allowing for a detailed description of the mutational and chromosomal landscapes. This in turn has increased our understanding of common driver events in thyroid tumorigenesis as well as highlighted markers of diagnostic, prognostic, and therapeutic value in the clinical setting.In the study outlined above, the authors amassed evidence for the progressive model, as thyroid tumors were clonally linked, further supporting the theory that thyroid cancer dedifferentiation in this case was a chained genetic event rather than synchronous manifestations of tumors arising separately by random chance. Moreover, it seems tempting to speculate that a proportion of ATCs develop as a consequence of aberrant DNA repair mechanisms due to somatic inactivation of MMR genes in pre-existing WDTCs, in turn leading to hypermutability and the generation of multiple tumor sub-clones, of which some might give rise to an undifferentiated phenotype ,6. The advent of molecular testing has helped identify subset of tumors with potentially targetable mutations, not least mutations in BRAF, mTOR, and ERBB2. Even though partial responses have been noted, exceedingly few patients survive once the tumor spreads to distant sites,14. The notion that a proportion of ATCs exhibit MMR deficiency should therefore not be overlooked, as MMR deficient tumors could mandate specific therapeutic considerations, and as the hypermutability in turn might give rise to additional targetable mutations[44]. Especially the promising value of immune checkpoint inhibitors in subsets of ATCs should be evaluated against the MMR status of these tumors, as the hypermutability in MMR deficient ATCs in theory would generate a massive overrepresentation of “non-self” tumoral antigens, in turn priming the patient’s immune response. However, as most ATCs lack hypermutability and MMR deficiency, the identification of additional molecular mechanisms suitable for targeted therapies is still highly warranted for the majority of cases.ATCs are feared tumors in the clinical setting, not least due to the limited response to conventional treatment optionsTo conclude, ATC patient outcome is exceedingly poor when distant spread is evident. While conventional treatment options are largely ineffective, targeted molecular therapy has shown promising results in terms of prolonged survival even after disease dissemination. The knowledge that subsets of ATCs probably arise as a clonal evolution from DNA repair malfunctioning precursor tumors could potentially be of importance when considering adjuvant treatment options for this ill-fated patient category, not least through direct or indirect targeting of a defective MMR system.


The issuing of regulation schemes and the expanding health insurance coverage for virtual visits of internet hospitals would incentivize Chinese providers and patients to use virtual visits tremendously. China’s internet hospitals vary in sponsorship. However, little is known about patients’ intention to use virtual visits delivered by different sponsorship types of internet hospitals.The goal of the research is to examine patients’ intention to use virtual visits, as well as virtual visits delivered by different sponsorship types of internet hospitals. In addition, we will identify determinants of patients’ intention to use virtual visits, as well as intention to use virtual visits delivered by different sponsorship types of internet hospitals.A cross-sectional survey of 1653 participants was conducted in 3-tier hospitals in 3 cities with different income levels in May and June 2019. Binary logistic regression analysis was used to identify the factors that affect patients’ intention to use virtual visits. Multinomial logistic regression analysis was conducted to identify the determinants of the intention to use virtual visits delivered by different sponsorship types of internet hospitals .A total of 76.64% (1145/1494) of adult participants were online medical information seekers, and 87.06% (969/1113) of online medical information seekers had intention to use virtual visits. Public hospital–sponsored internet hospitals were the most prevalent ones among Chinese patients , followed by the provincial government internet hospital platform , digital health companies , medical e-commerce companies , private hospitals , and other companies . Gender, education, monthly income, and consumer type were significantly associated with the intention to use virtual visits. Gender, age, education, city income level, consumer type, and trust in the sponsor of a health website were significantly associated with the patient’s intention to use virtual visits delivered by 3 different sponsorship types of internet hospitals.Chinese patients who were online medical information seekers had high intention to use virtual visits and had different intentions to use virtual visits delivered by different sponsorship types of internet hospitals. Public hospitals, the government, and digital health companies were the top 3 sponsorship types of internet hospitals that patients had intention to use. Trust in a health website sponsor significantly influenced the patient’s intention to use virtual visits delivered by different sponsorship types of internet hospitals. Gender, education, and consumer type were the factors significantly associated with both the intention to use virtual visits and the intention to use virtual visits delivered by different sponsorship types of internet hospitals. Virtual consultation and virtual visit are two primary types of services delivered by internet hospitals in China. An internet hospital is, to a large extent, an equivalent of direct-to-consumer telemedicine. China’s first internet hospital officially opened in 2014 , and theIn addition to the difference in scope of service, virtual visit is also different from consultation in health insurance coverage. Previous research has suggested that health insurance coverage is a significant factor that affects the use of virtual visits . FollowiInternet hospitals vary in sponsors. There are primarily brick-and-mortar hospital-sponsored and enterprise-sponsored internet hospitals in China’s direct-to-consumer telemedicine market . RecentlWe conducted a cross-sectional survey in Zhejiang province to examine patients’ intention to use virtual visits and their intention to use virtual visits delivered by different sponsorship types of internet hospitals and identify the factors that affect patients’ intention to use virtual visits. Zhejiang takes a leading role in China’s internet hospital industry development because it has the first licensed enterprise-sponsored internet hospital (WeDoctor Group)  and the When our survey was conducted, Zhejiang was the first and only province in China that delivered direct-to-consumer telemedicine services including virtual visits for its residents via its provincial internet hospital platform. Only after the outbreak of COVID-19 did other provincial governments start to deliver direct-to-consumer telemedicine service to their residents; however, the majority of services were specially designed free virtual consultations to contain the COVID-19 epidemic. Zhejiang is currently still the leading province to deliver virtual visits by pooling all local health care resources via its provincial internet hospital platform. Therefore, a survey on residents in Zhejiang Province can provide prospective insights for research questions on Chinese consumers’ intention to use virtual visits delivered by hospital-sponsored, enterprise-sponsored, and government-sponsored internet hospitals.As two primary service types of internet hospitals, virtual visit and virtual consultation are different from each other in service scope and health insurance coverage. However, there is hardly a clear division between virtual consultation and virtual visit in the current knowledge of internet hospital literature. Previous studies have examined either patients’ intention to use an internet hospital ,13 or a Systematic reviews have revealed that the technology acceptance model (TAM) was the most commonly used model to examine users’ acceptance of health information technologies including telemedicine -21. The Websites, apps and WeChat public accounts are 3 primary modalities through which Chinese providers deliver direct-to-consumer online health information and services. Health websites and apps in China usually cover provisions of health/medical information and knowledge, in-person visit online appointment, result tracing of laboratory and diagnostic imaging tests, and consultation, etc. For some health websites that have been licensed to deliver internet hospital services, the internet hospital, especially a virtual visit, is also an essential part of these health websites. However, among all types of online health information services, Chinese patients had the highest awareness and use of in-person visit appointments and medical fee payment online . TherefoThe classification of health websites is also different from that of internet hospitals in the current literature. The website owner/sponsor has been identified as one of the most widely reported indicators consumers applied to evaluate the quality of online health information ,27. InteWith reference to the Health Information National Trends Survey (HINTS) of the US National Cancer Institute, we made some modifications in accordance with China’s context and research questions to design our survey questionnaire .We conducted a cross-sectional survey using stratified sampling in 3 cities of different income levels in Zhejiang Province, China. According to Zhejiang Statistical Yearbook 2019 , 5 citieA pretest with a sample of 39 participants was conducted in April 2019. Based on China’s 3-tier health care delivery system, hospitals are designated as primary, secondary , and tertiary institutions . This study conducted an in-person structured questionnaire survey in 5 different types of hospitals  in May and June 2019. A total of 1653 participants were then randomly surveyed in the outpatient center of each hospital, and health practitioners were excluded from the survey. Hangzhou was the high-income city (n=871), Jinhua was the medium-income city (n=401), and Lishui was the low-income level city (n=381).Sociodemographic characteristics of gender, age, education level, and marital status were included in the analysis. Monthly income level  was included. Participants were asked to rate their current health status using a 5-point Likert scale ranging from very good (1) to very poor (5).According to Internet Development Report of Zhejiang Province 2019, 80.9% of its residents had access to the internet . TherefoWe measured the trust in health website sponsorship types among Chinese patients by asking: “Which type of health website do you perceive as the most trustworthy source of medical information?” Websites of medical schools/universities and academic institutions do not provide direct-to-consumer online medical information and services for lay persons; consequently, there are no educational health websites in China. Based on China’s context, we categorized health websites into 4 website sponsorship types: governmental, commercial , organizational , and personal . The option of other was included in case there were some participants who had no prior experience and knowledge of health websites. Rice et al  referredWe measured the intention to use virtual visits of internet hospitals by asking: “The government has issued guidelines on the development of internet hospitals. Virtual visits of internet hospitals enable physicians to diagnose, treat, and prescribe for some common conditions and chronic diseases for non–first-visits and deliver the prescription right to your door. Do you have the intention to use the virtual visit?”We measured the intention to use virtual visits delivered by different sponsorship types of internet hospitals by asking: “Which type of internet hospital do you have the highest intention to use regarding a virtual visit?” There were the facts regarding sponsorship types of internet hospitals in China: (1) hospital sponsors included public hospitals and private hospitals; enterprise sponsors included digital health companies, internet tech companies, medical e-commerce companies, medical informatics companies, health management companies, hospital management companies, pharmaceutical companies, insurance companies, medical equipment companies, etc  and (2) Stata 12.0 software (StataCorp LLC) was used to conduct statistical analyses. Descriptive statistics identified sociodemographic characteristics of the final sample, proportions of participants who had the intention to use virtual visits, and proportions of participants who had the intention to use virtual visits delivered by different sponsorship types of internet hospitals.A systematic review of end user acceptance of telemedicine use  found thHealth information seeking was the patient’s most frequently used eHealth or mobile health  activity , and heaAmong adult participants, 76.64% (1145/1494) were online medical information seekers. Among medical information seekers, those who did not answer the question of intention to use were excluded. The binary logistic regression result of intention to use virtual visits of internet hospitals is presented in Multinomial logistic regression was used to identify the determinants of intention to use virtual visits delivered by 3 major different sponsorship types of internet hospitals . The final sample for the multinomial logistic regression analysis was 894. Using hospital-sponsored internet hospitals as the base outcome, we had following results. Females were more likely to have intention to use virtual visits delivered by the governmental internet hospital platform and less likely to have intention to use visits delivered by enterprise-sponsored internet hospitals. Participants aged 30 to 40 years were more likely than those aged 18 to 29 years to have intention to use the governmental internet hospital platform. Participants with higher education levels were less likely to have intention to use virtual visits delivered by enterprise-sponsored internet hospitals. Participants in a medium-income city (Jinhua) were less likely than those in a high-income city (Hangzhou) to have intention to use virtual visits delivered by both enterprise-sponsored internet hospitals and governmental internet hospital platform. Consumers of secondary A, tertiary B, and tertiary A hospitals were less likely than consumers of primary hospitals to have intention to use enterprise-sponsored internet hospitals. Participants who trusted commercial health websites most were more likely than those trusted governmental health websites to have intention to use virtual visits delivered by enterprise-sponsored internet hospitals rather than visits delivered by hospital-sponsored ones. Compared to participants who trusted in governmental health websites, participants who trusted all other sponsorship types of health websites were less likely to use virtual visits delivered by the governmental internet hospital platform.Chinese patients who were online medical information seekers had a high intention to use virtual visits of internet hospitals. Gender, education, monthly income, and consumer type were significantly associated with the intention to use virtual visits. Patients had different intentions to use virtual visits delivered by different sponsorship types of internet hospitals, in which the public hospital–sponsored one was the most prevalent one, followed by the government, digital health companies, medical e-commerce companies, private hospitals, and other companies. Gender, age, education, city income level, consumer type, and trust in health website sponsor were significantly associated with the patient’s intention to use virtual visits delivered by 3 different sponsorship types of internet hospitals .This study, to our knowledge, was the first survey that examined the intention to use virtual visits delivered by different sponsorship types of internet hospitals among patients in China. Previous studies have examined the intention to use internet hospitals in China; however, most of them have not specifically examined the intention to use virtual visits of internet hospitals ,13. ThisFindings of this study have confirmed Hypothesis 2 that patients had different intentions to use virtual visits delivered by different sponsorship types of internet hospitals, as well as Hypothesis 1 that trust in sponsor was the significant determinant of the patient’s intention to use virtual visits delivered by different sponsorship types of internet hospitals. Public hospitals and the government were the top 2 sponsors of internet hospitals in which patients had high intention to use virtual visits, which was consistent with previous studies that found patients tended to trust health websites sponsored by hospitals, universities, government agencies, and well-known nonprofit organizations ,41. EnteThree different categories of internet hospitals  varied in target customer, motivation, and online health care resource allocation. The government-sponsored internet hospital aims to deliver internet hospital services to all its local residents by pooling almost all its regional public medical institutions. Hospital-sponsored internet hospitals usually confine the health care resource to the individual hospital itself or the medical alliance brought by the integrated delivery system which often includes public hospitals and primary health care institutions . EnterprTrust  has 2 primary components: trustworthiness (perceived motivation) and expertise (perceived ability) to provide accurate and truthful information . PatientPatients had higher intention to use virtual visits delivered by digital health company–sponsored internet hospitals (12.98%) than medical e-commerce company-sponsored ones 5.37%) and other companies (0.67%), which was in accordance with the fact that digital health companies played a dominant place in the enterprise-sponsored internet hospitals (44%) [.37% and The findings on the association of sociodemographic variables and the intention to use telemedicine remained mixed. This study identified that gender, education, income, and consumer type were the factors significantly associated with the intention to use virtual visits. Most previous studies also found that patients who were more willing to use telemedicine tended to have a higher education level ,47,48. TThis study indicated that gender, education, and consumer type were the factors significantly associated with both the intention to use virtual visits and the intention to use virtual visits delivered by different sponsorship types of internet hospitals. Patients with the characteristics of being female, higher education level, tertiary A and B hospital, and secondary A hospital consumers were more likely to have intention to use virtual visits delivered by hospital-sponsored internet hospitals other than enterprise-sponsored ones. These groups of patients had higher demand for high-quality care in which hospital-sponsored internet hospitals were perceived as a better deliverer than enterprise-sponsored ones.Females were more likely than males to prefer the government-sponsored internet hospitals to hospital-sponsored ones, as on one hand the government-sponsored one was more convenient for females (the primary family caregiver) because it pooled the regional public hospitals together; on the other hand, female Chinese were more likely to trust in governments . PatientCompared to enterprise-sponsored and government-sponsored internet hospitals, patients in medium-income cities were more likely than those in high-income cities to have intention to use virtual visits delivered by hospital-sponsored ones. Zhejiang Province has done a good job in market penetration, since there are quite a few internet hospitals at municipal level and even at county level . The morThis study has several limitations. First, this study specifically examined online medical information seekers’ intention to use virtual visits delivered by internet hospitals, which might overestimate the whole group of patients’ intentions to use. Second, there are urban-rural divides at various aspects in China. The survey was conducted in cities of Zhejiang Province. As rural population was not included in this survey, the findings of this study only revealed urban patients’ behavioral intention toward virtual visits. We will extend our study by conducting the survey on the whole population  in the future.Third, Zhejiang provincial internet hospital platform was initially launched on Alipay  in January 2019. Our survey was conducted in May and June 2019, and in July 2019, the Zhejiang provincial internet hospital platform was transferred to the all-in-one digital provincial government website/app. This transfer might have some effects on patients’ intention to use the government-sponsored internet hospital platform.Fourth, this study was conducted before the outbreak of COVID-19, which may have been greatly different from the current situation. The outbreak of COVID-19 has brought tremendous growth of internet hospitals in China and has drastically increased the awareness of virtual visits. The rapid increase of awareness would greatly increase the patient’s intention to use and actual use of virtual visits. We will follow and further the study by continuous survey on patients’ behavioral beliefs, intention to use, and actual use of internet hospitals to measure the impacts of drastic expansion of different sponsorship types of internet hospitals  brought by the outbreak of COVID-19.This study has demonstrated that virtual visits differ from virtual consultations in both service scope and health insurance coverage. The virtual visit market is an emerging market in China. This study has implied that different sponsorship types of internet hospitals  have different motivations, target consumers, and online health care resource allocation. This study found that Chinese patients who were online medical information seekers had high intention to use virtual visits. Public hospitals, the government, and digital health enterprises were the top 3 sponsorship types of internet hospitals in which patients had intention to use their virtual visit services.This study revealed that trust in a health website sponsor significantly influenced the patient’s intention to use virtual visits delivered by different types of internet hospitals, which extended the current knowledge regarding the impact of trust on adoption of direct-to-consumer telemedicine service. This study implied that internet hospitals should pay more attention to consumers with characteristics of being female and a tertiary hospital consumer with higher education level and high income level when developing the virtual visit market. It also implied that different sponsorship types of internet hospitals had different target consumers for virtual visit service. Hospital-sponsored internet hospitals had an advantage over the government-sponsored and enterprise-sponsored ones when developing the virtual visit market in medium-income cities.


Next-generation sequencing (NGS) technology has revolutionized sequence-based research. In recent years, high-throughput sequencing has become the method of choice in studying the toxicity of chemical agents through observing and measuring changes in transcript levels. Engineered nanomaterial (ENM)-toxicity has become a major field of research and has adopted microarray and newer RNA-Seq methods. Recently, nanotechnology has become a promising tool in the diagnosis and treatment of several diseases in humans. However, due to their high stability, they are likely capable of remaining in the body and environment for long periods of time. Their mechanisms of toxicity and long-lasting effects on our health is still poorly understood. This review explores the effects of three ENMs including carbon nanotubes (CNTs), quantum dots (QDs), and Ag nanoparticles (AgNPs) by cross examining publications on transcriptomic changes induced by these nanomaterials. Sequence based high-throughput technology has made it possible to observe gene expression changes and is quickly becoming the preferred choice in toxicological studies through identifying the modes of action (MOA) or the mechanisms of toxicity of certain chemicals and materials. These technologies are extremely sensitive and capable of whole-transcriptome sequencing and can quantify gene expression alterations by measuring RNA transcripts. The most accurate technique, RNA-Seq, has been around for more than a decade and can detect the slightest changes in gene expression with unprecedented accuracy . It is qENMs include a wide variety of nano-sized particles with incredibly small size ranges of 1–100 nm  that posIt has been commonly established in ENM-toxicity studies that their physiochemical properties contribute to their toxicity. The high diversity in physiochemical properties amongst ENMs has made characterizing their modes of action or identifying their individual mechanisms of toxicity in biological systems exceedingly difficult ,10. In aIn this review, we attempt to examine the toxicity of three ENMs including AgNPs, carbon nanotubes (CNTs), and QDs. AgNPs and CNTs were selected because silver is the most common nanomaterial used in products, followed closely by carbon-based nanomaterials . AdditioDNA microarray and RNA-Seq are the best methods for whole transcriptome gene expression profiling in toxicogenomic studies ,14. DNA The limitations of DNA microarray are apparent when target sequences are composed of unknown genomic sequences. This method only works by using nucleic acid probes of known sequences that are covalently bound to a slide. Therefore, microarray techniques are only as accurate as the bioinformatic data available on an organism’s genome . In compRNA-Seq is clearly the superior technique for gene expression profiling, however, DNA microarray is still valid and implemented in gene expression investigations. Due to the rapid advancements in software, RNA-Seq will eventually become more cost effective. Currently, the use of DNA microarray and RNA-Seq in evaluating ENM-toxicity is not very common, but is becoming more frequent, RNA-Seq more so than DNA microarray. Notably, DNA microarray techniques will not become obsolete anytime soon partly due to their clinical applications in disease diagnostics . Soon, RDue to the evident advantages of using RNA-Seq in identifying changes in gene expression, toxicogenomic techniques have become a useful tool for assessing risk to human health . These tCNTs are a highly versatile class of ENMs that have a variety of uses ranging from high performance batteries and touchscreens to drug delivery systems  5]. The. The5]. Previous studies have shown that exposure to CNTs can have toxic effects through the generation of oxidative stress or by inducing inflammation . MWCNT aEscherichia coli (E. coli) . As. AsGSS, p53, p21, Cyt C, Bid, Bax, and Bak; modification of signaling molecule expression related to cell survival, viability, and cytotoxicity were also observed , in cervical cancer cells (HeLa) by Yuan et al. The combined treatments increased the expression of pro-apoptotic genes like observed  74]. NG. NGp53, The study of AgNPs in human tissue is crucial to discovering the strengths/limitations of their application to human cancer. Opposed to their anti-cancer potential, recent evidence has shown that AgNPs alter genetic expression to increase susceptibility to carcinogens  76]. Ou. Ou76]. In conclusion, the use of AgNPs has greatly impacted antibacterial, antifungal, and anticancer treatment methods. This review only touches the surface of data that has been reported, but shows evidence concluding that AgNPs are effective in the alteration of gene expression among species. While its properties are promising, careful consideration is still needed in reference to their toxicity within mammalian cells. Going forward, more research is needed to understand what levels of AgNPs are safe for human consumption and ways to harness their toxicity so that they can continue to be an effective treatment against pathogenesis.Toxicogenomic techniques such as DNA microarray and RNA-seq could be valuable tools for assessing toxicity. However, RNA-seq provides more precise coverage of the transcriptome of a cell . Due to


In Europe, liver cirrhosis represents the fourth-most common cause of death, being responsible for 170,000 deaths and 5500 liver transplantations per year. The main driver of its decompensation is portal hypertension, whose progression radically changes the prognosis of affected patients. Transjugular intrahepatic portosystemic shunt (TIPS) is one of the main therapeutic strategies for these patients as it reverts portal hypertension, thus improving survival. However, the coexistence of portal hypertension and pulmonary hypertension or heart failure is considered a contraindication to TIPS. Nevertheless, in the latest guidelines, the definition of heart failure has not been specified. It is unclear whether the contraindication concerns the presence of clinical signs and symptoms of heart failure or hemodynamic changes in the right heart-pulmonary circulation. Moreover, data about induced right heart volume overload after TIPS and the potential development of heart failure and pulmonary hypertension is currently scanty and controversial. In this article we revise this issue in finding predictors of cardiac performance after TIPS procedure. Performing a fluid challenge during right heart catheterization might be a promising expedient to test the adaptation of the right ventricle to a sudden increase in preload in the first few months after TIPS. This test may unmask a potential cardiac inability to sustain the hemodynamic load after TIPS, allowing for a clearer definition of heart failure and, consequently, a more robust indication to TIPS. In Europe, liver cirrhosis is the cause of 5500 liver transplantations per year and it constitutes the fourth most-common cause of death, resulting in 170,000 deaths per year , which ePortal hypertension is a syndrome caused both by an increased resistance in the portal intrahepatic circulation and by increased splanchnic blood flow. In healthy liver, the pressure difference between the portal and the hepatic veins does not usually exceed 5 mmHg. Portal hypertension is defined by a gradient greater than 6 mm Hg, even if clinical complications do not seem to occur until the gradient exceeds 10–12 mmHg . The preThe introduction of transjugular intrahepatic portosystemic shunt (TIPS) in clinical practice represented, in the last 20 years, a step forward for treatment improvement for patients with untreatable complications of portal hypertension.TIPS works as a shunt between the systemic and the portal circulation. It is obtained by percutaneously connecting an intra-parenchymal branch of the portal vein with a hepatic vein through a low-resistance conduit, embraced by a self-expandable metal stent. Thus, TIPS reduces the portal–caval pressure gradient. The current criteria for TIPS placement have been enlarged . ClinicaThe impact of TIPS on pulmonary hemodynamics is still poorly discussed in the scientific literature. Creation of a TIPS may critically influence the pulmonary circulation. Blood shifts from the splanchnic circulation to the systemic circulation, thus increasing cardiac preload. Such changes can be clearly evidenced utilizing invasive hemodynamic techniques through right heart catheterization. Indeed, blood shunting results in an increase in cardiac output (CO) and pulmonary artery pressure and a decrease in systemic vascular resistances . A recenFinally, it was shown that volume overload induced by TIPS placement may be associated with increased PAWP and, consequently, postcapillary pulmonary hypertension (group II WHO classification) ,22,23,24Cardiac assessment for elective TIPS insertion, according to guidelines , includeTIPS is contraindicated in patients with severe left ventricular dysfunction or severe pulmonary hypertension  . HoweverOn the other hand, a Consensus Conference on TIPS management published in 2017 proposed hemodynamic and echocardiographic thresholds contraindicating TIPS positioning in case of pulmonary hypertension . These thresholds, indicative of severe pulmonary hypertension (PH), would be predictive of cardiac dysfunction after TIPS. However, TIPS can be performed in mild PH (mPAP at RHC between 25 and 35 mmHg) . These dEven if there is a growing awareness of the importance of a correct cardiac evaluation, since blood shunting after TIPS is responsible for a consistent increase in right atrial pressure (RAP) and cardiac output (CO), predictive studies of adverse cardiac outcomes after TIPS are currently scarce and controversial . A studyThe proportion of patients with possible cardiac complications may further increase in the near future as patients with cirrhosis of metabolic origin are increasing in prevalence. These patients show concomitant diabetes, arterial hypertension, and/or high HDL cholesterol or triglycerides concentration in blood, and increased cardiovascular risk. For this reason, an accurate cardiac assessment becomes crucial in their evaluation before TIPS placement . The corUnfortunately, long-term changes in the pulmonary circulation-heart unit induced by volume overload after TIPS placement are currently overlooked in clinical practice ,30,31,36The potential impact of TIPS placement on the pulmonary circulation offers the opportunity to better clarify the correct balance of its clinical indication.According to current guidelines/consensus, increased right and/or left ventricular diastolic pressure would allow for TIPS contraindication even in the absence of structural heart diseases ,14. IndeInvasive hemodynamic assessment would also allow us to clearly identify increased pulmonary pressure, which represents a second important contraindication for TIPS placement. The correct measurement of PAWP allows definitively to identify precapillary pulmonary hypertension  from postcapillary pulmonary pressure  ,39.Additionally, RHC assessment gives us the unique opportunity to test the potential consequences of TIPS-derived volume overload on the pulmonary circulation-heart unit.Considering such important clinical consequences of TIPS placement, a fluid challenge during initial RHC could be a promising tool to correctly evaluate the potential hemodynamic changes induced in the pulmonary circulation-heart unit in patient candidates for TIPS.In recent years, indeed, many studies have shown that a fluid challenge with rapid saline infusion during RHC was useful to correctly identify hidden left heart failure related to left ventricular diastolic dysfunction in the absence of structural heart diseases . Fluid cThe fluid challenge test during initial RHC could help clinicians in the difficult task of deciding on TIPS indication in a different clinical setting. A relevant cut-off value of RAP increase during rapid saline infusion would probably improve our understanding for the correct balance between indication and contraindications to TIPS placement and provide essential information on outcomes for future decision-making processes.Further investigations on pulmonary circulation-heart unit behaviors and their influence on the outcome of TIPS are still required.


Disrupted interoceptive processes are present in a range of psychiatric conditions, and there is a small but growing body of research on the role of interoception in obsessive-compulsive disorder (OCD). In this review, we outline dimensions of interoception and review current literature on the processing of internal bodily sensations within OCD. Investigations in OCD utilizing objective measures of interoception are limited and results mixed, however, the subjective experience of internal bodily sensations appears to be atypical and relate to specific patterns of symptom dimensions. Further, neuroimaging investigations suggest that interoception is related to core features of OCD, particularly sensory phenomena and disgust. Interoception is discussed in the context of treatment by presenting an overview of existing interventions and suggesting how modifications aimed at better targeting interoceptive processes could serve to optimize outcomes. Interoception represents a promising direction for multi-method research in OCD, which we expect, will prove useful for improving current interventions and identifying new treatment targets. OCD affects 1–3% of adults , 3. It iSeminal conceptualizations of OCD that emphasize the role of cognitions and fear in the development and maintenance of symptoms are particularly relevant for obsessions and compulsions related to preventing or avoiding a feared outcome or bad event . Indeed, traditional anxiety-based models form the foundation for evidence-based CBT interventions such as exposure and response prevention ExRP; –11. HoweA growing body of research has begun to investigate the role of sensory processing in OCD, including the processing of internal, or interoceptive stimuli (and the focus of this paper) and external (exteroceptive) stimuli  is isemotioals . In TS,n = 19–21) combined with slight differences in task design  could have contributed to inconsistent findings.Limited research demonstrates that interoceptive processes may relate to premonitory urges in adults with Tourette Syndrome. Rae et al.  found thIt has been suggested that urges preceding repetitive behaviors in OCD and TD may be phenomenologically similar to “urges-for-action,” which are everyday sensations that motivate behaviors such as blinking or scratching , 138. A Disgust is a basic emotion that functions to motivate avoidance of potentially harmful stimuli that could cause disease , 144. ItNot surprisingly, in both non-clinical and OCD samples, evidence reliably demonstrates a connection involving disgust-proneness with contamination symptoms and behavioral avoidance –157. FurCompared to controls, individuals with OCD showed greater activity in left and right insula when viewing disgust-inducing images, but did not show different patterns of neural activity when viewing fear/threat-inducing images –166. As To date, self-report and neuroimaging investigations provide the most compelling evidence for interoceptive differences in patients with OCD. Further, interoception may be more relevant to specific clinical presentations, including individuals with symptoms of symmetry/ordering motivated by sensory phenomena or contamination/washing driven by visceral feelings of disgust. Beyond self-report data, neuroimaging investigations demonstrate the involvement of key interoceptive regions like the insula in the pathophysiology of sensory phenomena, urges-for-action, and disgust. Given this, looking at interoception and related core OCD phenomena in the context of treatment could provide valuable insights necessary for improving therapeutic outcomes.Cognitive behavioral therapy (CBT) with exposure and response prevention (ExRP) is the gold standard treatment intervention for OCD . Responsin vivo) or mental stimuli  that elicit fear, rather than target sensory-based symptoms. Still, there is evidence suggesting that patients experiencing sensory phenomena derive greater clinical benefit from ExRP when it is optimized to specifically target those symptoms  including the tricyclic antidepressant clomipramine, and selective SRIs are considered a first line treatment for OCD , 187. HoDeep brain stimulation (DBS) involves the electrical stimulation of specific brain areas through implantation of electrodes. Most commonly used in the treatment of movement disorders such as Parkinson's disease, DBS is a FDA-approved intervention for treatment-refractory OCD , 197. ElTranscranial magnetic stimulation (TMS) is a non-invasive neuromodulation technique that involves placing a magnetic coil on the scalp that generates a brief and high-intensity magnetic field that excites or inhibits a part of the brain under the coil , 203. TMn = 5) or Tourette syndrome (n = 3) . An investigation applying inhibitory repetitive TMS over the bilateral SMA area for 10 daily sessions in a small sample of individuals with treatment-resistant OCD ( (n = 3)  found th (n = 3) –215, wit (n = 3) . A recen (n = 3) . In this (n = 3) .Even though the bulk of existing studies using TMS in OCD did not specifically evaluate changes in interoceptive processes, existing findings indicate that regions that are important in interoception could be indirectly (in the case of insula) or directly (in the case of sensorimotor regions) modulated by TMS . A recenBiofeedback generally involves measuring one's own physiological state and feeding the information back in real-time via visual or auditory or tactile feedback so that the individual can learn to modulate the physiological processes that are usually otherwise involuntary , 220. BiReal-time fMRI neurofeedback, another personalized approach, involves analyzing BOLD activity in real time as fMRI data is collected, and presenting information about neural activity in specific regions to the individual to guide modulation or self-regulation . NeurofeDespite increased understanding in the pathophysiology of OCD, current mainstay treatments have largely remained unchanged over the past 30 years. The clinical heterogeneity of a significant number of patients has not been fully accounted for by traditional anxiety-based models, thus prompting more research into the processing of internal sensations. Interoception presents itself as a promising target for OCD research given the established theoretical framework and measurable behavioral and biological correlates . Indeed,LB, ES, and GE contributed to the conception of the review. LB and AB searched and reviewed relevant literature. LB wrote the first draft of the manuscript. GE, ES, and KC wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Infection with certain types of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) viruses, known as tumor viruses or oncogenic viruses, can lead to cancer. Studies performed in 1911 on the Rous sarcoma virus (RSV), a retrovirus that induces cancer in chickens, opened the field of tumor virology, indicating that some cancers could be triggered by specific types of viruses. Since then, extensive investigations have been conducted on tumor viruses, leading to significant findings in cancer research. Moreover, these studies have unlocked new gates for discovering strategies to prevent or manage infections with tumor viruses. Therefore, this editorial paper introduces the Special Issue “Recent Progress in Tumor Virology Research”, which has been organized to collect recent studies on tumor viruses and their linked malignancies. The Special Issue has covered interesting research papers on various tumor viruses and their associated malignancies such as human papillomavirus (HPV), canine papillomavirus (CPV), Epstein–Barr virus (EBV), a novel identified retrovirus Gunnison’s prairie dog retrovirus (GPDRV), and Merkel cell polyomavirus (MCPyV). Two topical review articles on promising therapeutic strategies for oncogenic herpesviruses and their connected cancers and HPV-associated tumors were also included. The outcome of this Special Issue might improve our understanding of the full map of the interactions between tumor-inducing viruses and the infected hosts, guiding us to finding innovative approaches to combat these viral infections and their generated tumors.There is no doubt that infections with tumor viruses can cause serious health complications to both humans and animals. Inducing tumors is the most significant health problem reported with these viruses. Tumor viruses are categorized into two groups based on the type of nucleic acid, DNA viruses and RNA viruses ,2. The SMultiple research groups have performed well-designed experiments on HPV, the most frequent sexually transmitted virus in the world, and its connected malignancies. Ikegami et al.  developeCPV is a virus that belongs to the family of papillomaviruses with the ability to generate squamous cell carcinomas (SCCs) in dogs. The whole genomes of two CPV type 9 (CPV9) strains from recurrent SCCs isolated from benign or malignant skin lesions of two dogs were successfully analyzed by Chang et al. . The obtEBV, a gamma-herpesvirus , is one of the best-examined examples of a cancer-inducing virus . In a stButler et al.  successfKMT2D [MCPyV, a human oncogenic virus, has been observed to be accountable for the development of Merkel cell carcinoma (MCC), a lethal cancer of the skin, in humans. By employing a bioinformatics tool, MCPyV integration sites in whole-exome sequencing data from five MCC cases were entirely screened by an open access HPV Detector/Cancer-virus Detector tool. The study disclosed, for the first time, an integration that includes the tumor suppressor gene KMT2D .This Special Issue also involves two up-to-date review articles, representing various therapeutic approaches. Berberine, a natural alkaloid compound, was introduced as a promising natural drug for treating human oncogenic herpesvirus infections and their related cancers . Morgan Finally, all contributions that have been published in this Special Issue have delivered interesting ideas, techniques, and results, which provide a platform for other researchers to build upon.


Developmental origin of health and disease postulates that the footprints of early life exposure are followed as an endowment of risk for adult diseases. Epidemiological and experimental evidence suggest that an adverse fetal environment can affect the health of offspring throughout their lifetime. Exposure to endocrine disrupting chemicals (EDCs) during fetal development can affect the hormone system homeostasis, resulting in a broad spectrum of adverse health outcomes. In the present review, we have described the effect of prenatal EDCs exposure on cardio-metabolic-renal health, using the available epidemiological and experimental evidence. We also discuss the potential mechanisms of their action, which include epigenetic changes, hormonal imprinting, loss of energy homeostasis, and metabolic perturbations. The effect of prenatal EDCs exposure on cardio-metabolic-renal health, which is a complex condition of an altered biological landscape, can be further examined in the case of other environmental stressors with a similar mode of action. Environmental toxicants comprise a wide range of chemical agents released through natural or anthropogenic sources. They contaminate the abiotic components of the ecosystem and affect the health of the biotic components , elevated fasting glucose, and hypertension , and leptin levels in the exposed population  among children have been steadily increasing since the 1980s Baum, . A numbeDetectable bisphenol A levels were reported in a study on children correlated with increased levels of ACR , had increased β2-microglobulin (β2M) levels, suggesting some kidney damage  showed that cadmium exposure during childhood was inversely correlated with estimated glomerular filtration rate (eGFR), which can adversely affect kidney function  cause an estimated 17.9 million deaths annually  and DES during the embryonic stage led to disrupted cardiovascular development . A study on apes, which were orally administered BPA during the gestation period, suggested its impact on the cardiovascular fitness of the developing fetus , histone acetyltransferase (kat6a), and histone acetylation  mouse model, perinatal exposure to BPA worsened atherosclerosis in adult male huPXR∙ApoE−/− offspring  60. At later-life stages (PND 200), both systolic and diastolic systemic arterial pressure and locomotor activity were reduced in adult rats perinatally exposed to DEHP , a perfluoroalkyl substance (PFASs), was associated with elevated systolic blood pressure, low-density lipoprotein cholesterol (LDL-C), and total cholesterol  mice are highly vulnerable to atherosclerosis. The male offspring of pregnant ApoE−/− mice, which were exposed to arsenic, showed accelerated atherosclerotic plaque and loss of endothelial cell function and vascular tone at 10 and 16 weeks of age phthalate exposure in RAW 264.7 cells was found to cause inflammation through sirtuins  of ERα . Prenatal exposure to EDCs could cause adverse metabolic alterations, which are closely linked with CKD and CVD. The possible unifying mechanism could involve fundamental changes in the hormonal and epigenetic imprints caused due to EDCs exposure during fetal development. This could permanently alter the baseline inflammatory state and affect adipocyte development in the offspring. All these factors could increase the overall risk for cardiovascular disease, diabetes, and CKD in offspring. Developmental exposure to EDCs may thus be imprinting a risk phenotype for overall cardio-metabolic-renal health  in later life, which needs to be studied further.RS and KK conducted the comprehensive literature review. RS, KK, and VSh wrote the manuscript. RT, HK, and VSh helped in formulation of tables and figure and revised the article for important intellectual content. VSr and VSh designed the concept of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns. Genomes display large variability in their nucleotide composition, including in the frequencies and quantity of certain nucleotide pairs such as CpG and TpA (termed dinucleotides where consecutive nucleotides are linked by a phosphate bond on the same DNA strand) . ComposiAs viruses are obligate intracellular pathogens, they are dependent on the cellular translational machinery and in some cases, such as herpesviruses, the transcriptional machinery. They are therefore subject to the same effects caused by genomic compositional bias. Nevertheless, such biases were shown to play an important role in cellular defences against viruses, while viruses also manipulate compositional biases within their own genomes to evade host defence mechanisms .Herpesviruses are double-stranded DNA viruses with relatively large genomes that are highly disseminated in nature and can be found in almost all vertebrates and some invertebrates. There are currently over 150 viruses described, including 8 clinically important human herpesviruses and several significant veterinary pathogens .Millions of years of co-evolution have shaped Herpesviruses into masters of immune evasion ,5. A cenIn this review, we will discuss the role of compositional genomic bias in herpesvirus genomes and how the targeting of viral RNA with high CpG dinucleotide frequencies by ZAP may have shaped the evolution of herpesvirus genomes.Nucleotide content can vary drastically between different species and within a genome. For example, the GC content in human genomes can vary between approximately 35% to 60% over a 100 kb region, with high GC content associated with transcriptionally active regions, whereas AT-rich regions tend to be associated with heterochromatic regions with low gene density . NucleotIn addition to biases in overall nucleotide content, dinucleotide bias occurs when pairs of nucleotides occur more or less frequently than would be expected based on the overall nucleotide content of a gene or genome. Among these, CpG dinucleotides have been most extensively studied. CpG dinucleotide frequencies refer to the expected number of CpG sequences within a gene or genome compared to the overall GC content. For example, plant and vertebrate genomes show significantly lower CpG frequencies than would be expected given their overall base composition ,13,14. TPrevious studies have demonstrated significant suppression of CpG and UpA/TpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses, which mimics suppression in mammalian genomes . ArtificThe role of ZAP as a host restriction factor has recently been comprehensively reviewed . BrieflyZAP was originally identified as a host restriction factor by screening a rat cDNA library for proteins that inhibited the replication of Moloney murine leukaemia retrovirus . ZAP hasThere is now considerable evidence that ZAP is responsible for targeting transcripts with high CpG content. In 2017, Takata et al. showed that knockdown of ZAP rescued the replication of HIV constructs with artificially increased CpG levels introduced through synonymous mutations . CLIP-seenv gene, but not within the rest of the genome .,47.34,47The lack of significant suppression of CpG dinucleotides in alpha-herpesviruses, such as HSV-1, suggests the evolution of ZAP-specific counter-measures, as the high CpG transcripts would potentially represent strong targets for ZAP. This is supported by a previous study that showed HSV-1 was able to replicate to wild-type levels despite expression of a ZAP construct, although the construct expressed a truncated version of rat ZAP, rather than full-length human ZAP . HSV-1 iHerpesvirus express their genes in a strict temporal manner; immediate-early transcription, followed by early and late transcription. For HCMV, this was further refined to seven temporal classes . The majThe lack of suppression of CpG dinucleotides in the HCMV genes expressed subsequent to IE1, suggests a loss of pressure to maintain low CpG levels in these genes, possibly due to the loss of ZAP targeting. HCMV effectively blocks IFN signalling early in infection, in part through the actions of IE1 and IE2, suppressing ZAPS induction. However, initial western blot analysis showed high levels of ZAP expression throughout HCMV infection of cells. This would be counterintuitive based on the CpG pattern of the virus, as ZAPS would be predicted to target early and late genes, effectively inhibiting virus replication. However, separation of cells by sorting into high and low infection levels based on viral GFP expression, showed that cells with productive infections had low levels of ZAP expression, while high levels of ZAP expression occur in abortively infected cells, indicating a population-specific pattern of ZAP expression missed by standard bulk western blot approaches. Rather than a ZAP specific phenomenon, this likely reflects a balance between the ability of the virus to successfully shut down the IFN response, versus the host cell mounting a response that is able to inhibit the progression of virus replication at an early stage.The precise mechanism by which ZAP inhibits HCMV replication is still unclear. A recent study identified transcripts from the UL4–UL6 region as specific targets of ZAP during infection with HCMV. Analysis of total and newly synthesised viral RNA showed accelerated expression of UL4–UL6 transcripts in ZAP knockout cells, while CLIPseq analysis identified ZAP binding sites within these transcripts. Why ZAP specifically targets these transcripts is unclear, as there appears to be a limited difference in dinucleotide content in UL4–UL6 transcripts compared to other HCMV transcripts, other than IE1, which has suppressed CpG levels. UL4–UL6 is among the most abundantly transcribed regions of the HCMV genome, which may explain the preferential binding of ZAP. It is also unclear why the virus has not evolved to evade targeting in this region. One possible explanation is that these transcripts act as a decoy target for ZAP, contributing to ZAP evasion, although further studies will be required to answer these questions .M2 and ORF64 [RTA), which encodes the viral protein necessary to initiate the lytic cycle, was also reported to inhibit ZAP activity, but as yet, the mechanism involved is unclear [Similar to RNA viruses and small DNA viruses, the majority of gamma-herpesviruses suppress CpG dinucleotides throughout their genomes, which would be predicted to evade targeting by ZAP. Despite this, murine gamma-herpesvirus-68 (MHV68) is susceptible to ZAP in tissue culture, with ZAP specifically targeting regions within nd ORF64 ,54. Bothnd ORF64 . ReplicaWhile suppression of CpG dinucleotides provides a clear fitness benefit to viruses due to evasion of ZAP targeting, questions remain as to why the majority of alpha and beta-herpesvirus genes maintain relatively high levels of CpG dinucleotides. While studies indicate that high CpG abundance alone does not predict ZAP binding, artificial increases in CpG frequencies in other viruses clearly leads to ZAP dependent attenuation. One could hypothesise that natural drift towards neutral CpG frequencies, based on overall GC content occurs due to lack of evolutionary pressure following successful evasion of ZAP. However, some alpha and beta-herpesviruses have low GC content  suggestiThis pressure may be due to the fundamental effects of nucleotide content on gene expression efficiency, especially in unspliced transcripts. As well as removing introns, splicing enhances nuclear export by facilitating the association of transcripts with the cellular RNA export machinery ,56. A reLimits on viral genome size, imparted by the physical constraints of capsid packaging, means the majority of herpesvirus genes are unspliced. This creates conflicting pressures on the nucleotide content of herpesvirus transcripts, with low CpG dinucleotide frequencies enabling ZAP evasion but potentially contributing to inefficient export and expression of unspliced transcripts. These forces may have shaped the evolution of herpesvirus sub-family genomes in different ways depending on the mechanisms employed to evade ZAP while maintaining efficient viral gene expression.However, additional factors may have also influenced the nucleotide content of herpes viruses. Epigenetic regulation of the viral genome has been implicated in the establishment, maintenance and reactivation of herpesviruses from latency . The sam6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs, N5-methylcytosine (m5C), was reported to enhance mRNA nuclear export through the recruitment of Aly, a nuclear mRNA export adaptor [Direct methylation of RNA can also regulate gene expression by modulating RNA splicing, export, stability and translation . While N adaptor . ChangesThe role of codon bias and codon pair bias in virus replication and gene expression was also investigated . SynonymGene expression is reliant on successive, interdependent post-transcriptional RNA processing events including splicing, polyadenylation, nuclear export and translation . The comBased on data from HSV-1, alpha-herpesviruses counter ZAP targeting immediately after infection, while beta-herpesviruses such as HCMV evade ZAP by suppressing CpG levels in the earliest transcripts before suppressing ZAP induction. Splicing of the immediate early genes ensures efficient expression despite suppressed CpG levels. A different scenario confronts gamma-herpesviruses, where CpG suppression occurs throughout the genome. While effectively evading potential targeting by ZAP, low CpG levels in unspliced genes could lead to inefficient expression, reducing viral fitness . To counBoth ICP27 and ORF57 directly recruit components of the TREX complex to unspliced viral transcripts, bypassing the requirement for splicing dependent recruitment and ensuring efficient translocation across the nuclear pore complex and into the cytoplasm ,75. Of tWhile all three proteins share a central functional role in facilitating the export of viral RNA, it is interesting to speculate whether these related viral proteins have evolved divergent RNA binding specificities in response to the characteristic dinucleotide content of each subfamily of herpesvirus. Multiple studies have attempted to determine consensus RNA binding motifs for ICP27 and ORF57. While binding sites with specific motifs were identified in a minority of viral and cellular transcripts , studiesEarly studies with ICP27 demonstrated a propensity for binding to G homopolymer substrates compared to other RNA homopolymers . FurtherThere are fewer studies directly addressing the binding specificities of ORF57. However, similar to ICP27, ORF57 binding preferences match the GC nucleotide content of the viral genome ,81. K15 Expression of ICP27 and its functional homologues may therefore have evolved, in part, to enable resolution of the conflicting pressures of evading ZAP detection, while maintaining efficient expression of unspliced transcripts. However, questions remain as to how this relates to the specific patterns of CpG dinucleotides in the different sub-families of herpesviruses. If ORF57 enables the efficient expression of unspliced transcripts with low CpG levels, why do the majority of alpha and beta herpesvirus transcripts maintain high CpG frequencies, potentially subjecting them to ZAP targeting? One possibility is that by maintaining a dinucleotide bias that is different from the host, it enables preferential export of viral transcripts over host transcripts through CpG specific binding by ICP27 or UL69 in the case of HSV-1 or HCMV. If this is the case, then gamma-herpesviruses, such as KSHV, must have developed an alternative method for differentiating between host and viral transcripts, as ORF57 would be unable to differentiate between host and viral transcripts based on CpG content. Alternatively, the different patterns of CpG dinucleotides may simply reflect divergent strategies of overcoming conflicting pressures of maintaining efficient viral gene expression while avoiding ZAP detection in the ancestral viruses of alpha, beta and gamma-herpesviruses.So far, studies aimed at understanding the binding preferences of ICP27 and ORF57 have not differentiated between GC content and CpG dinucleotide frequencies. While some alpha herpesviruses have much higher GC content compared to the host, others, such as Varicella Zoster Virus (VZV) have significantly lower GC content. Only CpG dinucleotide patterns are relatively uniform within the herpesvirus subfamilies. It would be interesting to compare the sequence binding preferences of ICP27 homologues from HSV-1 and VZV. Binding preferences of the ICP27 homologues would be different if GC content was the important factor, whereas they would be similar if based on CpG dinucleotide frequencies. Given only CpG dinucleotide frequencies are uniform across alpha-herpesviruses, and within herpesvirus sub-families in general, we would predict that binding preferences would be based on CpG frequencies, rather than GC content.These studies are consistent with an evolutionary paradigm in which conflicting pressures shape the nucleotide content of herpesviruses, resulting in characteristic and specific patterns of CpG frequency within each subfamily . Alpha-hBeta-herpesviruses do not trigger host shut off in infected cells and therefore need to contend with ZAP targeting immediately after infection. Our findings suggest HCMV, and other beta-herpesviruses, achieve this by suppressing CpG levels in the major immediate early genes, among the first transcripts expressed after infection. Immediate early proteins, including IE1, facilitate a block in IFN induction, reducing ZAPS expression. A caveat to this model is the potential antiviral role of ZAPL, which is constitutively expressed and would not be affected by a block in IFN signalling. However, data from our paper show reduced levels of both ZAPS and ZAPL in cells productively infected with HCMV, suggesting inhibition of ZAP expression may be playing a role .Gamma-herpesviruses have evolved low CpG frequencies throughout the genome, thereby reducing potential targets for ZAP.To maintain compact genomes, most herpesvirus genes are unspliced. While saving genomic space the general inefficiency in the expression of unspliced transcripts would potentially reduce the overall fitness of herpesviruses. Alpha- and beta-herpesviruses may have overcome this, to some extent, by maintaining higher CpG levels in the majority of their genes, which may increase the efficiency of expression, particularly in unspliced transcripts. All three herpesvirus subfamilies express RNA binding proteins that enhance gene expression by shuttling unspliced viral transcripts from the nucleus to the cytoplasm. This may be particularly important for gamma-herpesviruses, given the low CpG content of their genes. While not conclusive, studies also support specific adaptation of this family of RNA binding proteins to the unique nucleotide content of each herpesvirus subfamily, with ORF57 preferentially binding low GC transcripts, while ICP27 binds to transcripts with high GC content.A distinction between GC content and CpG frequencies can be difficult to disentangle. While GC content and CpG frequency are of course naturally linked, an increase in one does not always result in an increase in the other. For example, while there are clear distinctive patterns of CpG frequencies within herpesvirus subfamilies, no such pattern of GC content is apparent, suggesting a lack of clear biological relevance to GC content that would be linked to the evolution of the viruses. Further studies will be required to determine whether GC content or CpG frequencies play key roles in the expression of viral genes and binding specificities of ICP27 and its homologous.Finally, future studies could investigate whether altering the nucleotide composition of herpes viruses through synonymous mutations could be an effective platform for the generation of rational herpesvirus vaccine candidates or vectors. Modifying codon usage bias through the introduction of synonymous mutations could allow tuneable alterations to expression levels of viral target genes and levels of attenuation, as well as increasing the visibility of viruses to the host’s immune response while maintaining the natural antigenic profile of the virus .


In behavioral intervention research, taking a community-based participatory research approach enhances recruitment and retention while facilitating the transfer of research findings into social change. Successes with recruitment and retention are secondary to enacting fundamental principles of trust, reciprocity, cultural humility, empowerment, and respect. This presentation will describe a longitudinal clinical trial in a Southwest borderlands community, Oyendo Bien. The study was co-developed and implemented with community partnership throughout the research process. Dyads were recruited to participate in a community-delivered group education and support program addressing hearing loss for Spanish-speakers age 50+ years (n=132 participants randomized). We highlight the critical role that community health workers (promotoras) held as members of the research team. Furthermore, we describe an innovative approach for language mediation that integrates and empowers community participation. This presentation will include examples of lessons learned from the community in collaborating to conduct research in a way that truly serves.