Invasive lobular carcinoma (ILC) is a common but understudied breast cancer subtype. ILC is presumed to be a low-risk disease in part because nearly all ILCs contain the estrogen receptor (ER). However, we previously showed that ER has unique functions in ILC cells, including driving expression of the Wnt ligand WNT4. WNT4 signaling is required for ILC cell proliferation and survival, but the mechanisms and targets of WNT4 signaling in ILC is unknown. We found that WNT4 regulates mTOR signaling via S6 kinase, and controls levels of MCL-1 protein, ultimately regulating mitochondrial function and cellular metabolism. These findings offer new insight into a novel Wnt signaling pathway and identify new targets to inhibit WNT4 signaling as potential treatments against ILC cells.Invasive lobular carcinoma of the breast (ILC) is strongly estrogen-driven and represents a unique context for estrogen receptor (ER) signaling. In ILC, ER controls the expression of the Wnt ligand WNT4, which is critical for endocrine response and anti-estrogen resistance. However, signaling mediated by WNT4 is cell type- and tissue-specific, and has not been explored in ILC. We utilized reverse phase protein array (RPPA) to characterize ER and WNT4-driven signaling in ILC cells and identified that WNT4 mediates downstream mTOR signaling via phosphorylation of S6 Kinase. Additionally, ER and WNT4 control levels of MCL-1, which is associated with regulation of mitochondrial function. In this context, WNT4 knockdown led to decreased ATP production and increased mitochondrial fragmentation. WNT4 regulation of both mTOR signaling and MCL-1 were also observed in anti-estrogen resistant models of ILC. We identified that high WNT4 expression is associated with similar mTOR pathway activation in ILC and serous ovarian cancer tumors, suggesting that WNT4 signaling is active in multiple tumor types. The identified downstream pathways offer insight into WNT4 signaling and represent potential targets to overcome anti-estrogen resistance for patients with ILC. Invasive lobular carcinoma of the breast (ILC) is the second most common histologic subtype of breast cancer ,2,3. ClaWNT4 expression and novel WNT4 signaling pathways . RP. RPFASN reported  20]. Co. CoFASN reported B–D. WNT4reported E. siWNT4reported . These dWNT4 expression was associated with gene signatures for mTORC1 signaling and cellular metabolism  likely due to limited sample size of ILC with RPPA data . We expanded our RPPA analyses across other tumors from tissues that require WNT4 in development , lung adenocarcinoma , and serous ovarian cancer  presented differences in protein signaling by RPPA in WNT4-high tumors . These data in OvCa parallel our observations in ILC that WNT4 is associated with active PI3K/mTOR signaling. High WNT4 expression in OvCa was also associated with shorter overall survival  using the cBio Portal . In ILC , uterus ,46,47,48h tumors . Shared h tumors C. HoweveWNT4 expression. The Wnt ligand WNT4 plays a critical role in the normal mammary gland development, and similarly is required for endocrine response and resistance in ILC cells [WNT4 over-expression in ILC and in serous ovarian cancer tumors. These data provide new insight into a poorly understood Wnt signaling pathway and identify signaling pathways downstream of atypical intracellular WNT4 signaling [Clinical and laboratory data support that ILC represents a unique context for estrogen receptor signaling, which may be mediated in part by ER-induced LC cells . HoweverLC cells ,20,21. TOur data shows that ER drives mTOR signaling in ILC cells, with WNT4 serving as a necessary signaling partner for downstream signaling. Interestingly, rather than regulating phosphorylation of S6K at T389, we found WNT4 instead regulates phosphorylation at T421/S424. T421/S424 are part of a cluster of phosphorylation sites in the C-terminal auto-inhibitory domain of p70-S6K (pseudo-substrate inhibition), and their phosphorylation is necessary for S6K activation . The regWNT4 signaling is a novel regulatory mechanism for PI3K signaling via mTOR/p70-S6K. Activation of PI3K/Akt/mTOR signaling has been identified as a major feature of ILC, including increased pathway activity vs. matched IDC tumors, in three large-scale genomic analyses of ILC  ,58. ThesER:WNT4 regulation of MCL-1 and mitochondrial function suggest that WNT4 is critical for cellular metabolism in ILC cells. We observed that WNT4 knockdown leads to impaired ATP production and mitochondrial fragmentation, and both of these phenotypes precede the induction of cell death (~4d post-siWNT4 transfection ). This sWNT4 gene expression is elevated in OvCa tumors after neoadjuvant chemotherapy [The similar signaling pathways associated with WNT4 in ILC and OvCa may parallel the critical role of WNT4 in both tissues of origin, and related tumor biology. In addition to being required for mammary gland development, WNT4 is required for the development of the ovary and Mullerian tissues ,71,72. Aotherapy  and takeWNT4 signaling is a key mediator of endocrine response and resistance in ILC but given the dysfunction of β-catenin-dependent signaling in ILC and novel functions of WNT4 as an intracellular signaling molecule, WNT4 signaling pathways in ILC must be identified. In this study, our RPPA analyses identified mTOR signaling and MCL-1/mitochondrial function as two key downstream effectors of WNT4 in ILC. Parallel pathways associated with WNT4 were also identified in serous ovarian cancer, suggesting that WNT4 signaling is important in multiple tumor types. Future studies will determine how WNT4 is mechanistically linked to these targets, including identification of the WNT4 “receptor” in ILC cells. Furthering our understanding of WNT4 signaling will improve opportunities for precision treatment approaches by targeting WNT4 signaling for patients with ILC, or OvCa, and support the development of strategies to overcome anti-estrogen resistance for patients with ILC.ESR1-positive, and express low levels of ESR2 and GPER1 [2. Cell lines are authenticated annually via the University of Arizona Genetics Core cell line authentication service and confirmed to be mycoplasma negative every four months. Authenticated cells were in continuous culture <6 months.MDA MB 134VI (MM134) and SUM44PE (44PE) were maintained as described . MCF-7 and GPER1 . WNT4 ovnd GPER1  and cultnd GPER1 . All lin17β-Estradiol  and fulvestrant  were obtained from Tocris Bioscience  and dissolved in ethanol. Everolimus , PF-4708671 , ABT199 (cat#16233), ABT263 (cat#11500), WEHI539 , and A1210477  were obtained from Cayman Chemical  and dissolved in DMSO.WNT4 (M-008659-03-0005), human MCL1 (M-004501-08-0005). Details regarding validation of the specific effects of the WNT4 siRNA pool are previously described [siRNAs were reverse transfected using RNAiMAX (ThermoFisher) according to the manufacturer’s instructions. All constructs are siGENOME SMARTpool siRNAs , nontargeting pool #2 D-001206-14-05), human 6-14-05, g at 4 °C for 10′. Supernatant was collected and utilized for RPPA analyses. RPPA lysis buffer was made with 1% Triton X-100, 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, and 10% glycerol, supplemented with Halt protease and phosphatase inhibitor cocktail .Cells were hormone-deprived prior to reverse transfection with 10 nM siRNA as described above. Then, 24 h post-transfection, cells were treated with vehicle (0.01% EtOH) or 100 pM E2 and harvested 24 h later . Cells were lysed according to core facility instructions . BrieflRPPA analyses and data normalization were carried out by the MD Anderson Cancer Center Functional Proteomics Core Facility . Protein loading was kept consistent by mass across matched experiments, and standard methods were used to perform SDS-PAGE. Proteins were transferred onto PVDF membranes. Antibodies were used according to manufacturer’s recommendations: WNT4  for 30′ on ice. Cells were centrifuged at ~16,000× erations ); ERα  at 1 μg/mL in tris-NaCl buffer  at equivalent volume to lysate. Fluorescence (360 nm ex/460 nm em) was measured on a Bio-Tek  Synergy 2 microplate reader.Total double-stranded DNA was measured as a surrogate for total cell number by hypotonic lysis of cells in ultra-pure H15 primer (cat# C110A), random primers (cat# C118A), GoScript 5× reaction buffer (cat# A500D), 25mM MgCl2 (cat# A351H), 10mM dNTPs (cat# U1511), RNasin plus RNase inhibitor (cat# N261B) and GoScript reverse transcriptase (cat# A501D). qPCR reactions were performed with PowerUp SYBR Green Master Mix  on a QuantStudio 6 Flex real-time PCR system (ThermoFisher). Expression data were normalized to RPLP0. Primer sequences were published previously [RNA extractions were performed using the RNeasy Mini kit , mRNA was converted to cDNA on an Eppendorf Mastercycler Pro  and using Promega  reagents: oligo (dT)eviously ,20.For analysis of ATP per cell, cells were reverse transfected as above, and at the indicated timepoint, parallel plates were assessed for total dsDNA (above) and total ATP . Cell number by each method was normalized to a standard curve, and analyzed as a ratio of (cell number by ATP)/(cell number by dsDNA).MM134 cells were reverse transfected with siRNA as above, and 48 h later plated in technical duplicate per condition to 6 cm plates. Then, 24 h later , cells were collected by saline-EDTA wash and scraping, then fixed and pelleted in 2% glutaraldehyde, further processing was performed by the U. Colorado Anschutz Electron Microscopy Center. Pellets were re-suspended in 5% agarose that was allowed to harden, and then cut into small pieces for processing. Embedded cells were first rinsed three times in 0.1 M sodium cacodylate buffer (pH 7.4), then post-fixed in 1% osmium tetroxide for 1 h. After three washes in water, the cells were stained en bloc with 2% uranyl acetate for 1 h at 4 °C. Following dehydration through a graded ethanol series  and propylene oxide for 10 min each, the samples were infiltrated with LX112 resin. The samples were embedded and cured for 48 h at 60 °C in an oven. Ultra-thin sections (60 nm) were cut on a Reichert Ultracut S (Leica) from a small trapezoid positioned over the cells and were picked up on EMS copper mesh grids. Sections were imaged on a FEI Tecnai G2 transmission electron microscope  with an AMT digital camera . From collected images, mitochondrial dimensions were measured in ImageJ  as descrWNT4 expression (RNAseq) were selected based on the minimum z-score needed to exclude any low WNT4-expressing tumors. Statistical analyses were derived from cBio analysis tools, e.g., enrichments > protein > RPPA.TCGA data were accessed via the cBio portal in March–June 2019, provisional datasets were used for all analyses described. Ovarian cancer and ILC data were updated in June 2020 using the Firehose Legacy datasets. Z-score cutoffs for https://doi.org/10.17605/OSF.IO/7X8NG) upon publication, or upon request.Data associated with experiments herein will be available at an Open Science Framework repository  (https:/In models of invasive lobular carcinoma of the breast (ILC), ER-driven WNT4 signaling is critical for cell proliferation and survival, but downstream signaling mediated by ER:WNT4 is unknown. We identified mTOR signaling, via S6K, and MCL-1 as key targets of ER:WNT4 signaling in ILC cells, and that these targets converge upon control of mitochondrial dynamics and cellular metabolism. This study offers new insight into a novel Wnt signaling pathway in ILC cells, which underlies endocrine response and anti-estrogen resistance, and identifies potential targets to inhibit this pathway as new therapeutic strategies against ILC cells.


Rho GTPases are a family of small G-proteins of the Ras superfamily. The ubiquitously expressed Rho GTPases RhoA, Rac1, and Cdc42 are major regulators of the actin cytoskeleton, with essential functions in cell migration. However, Rho GTPases also regulate other processes than actin polymerization and are involved in the regulation of proliferation, differentiation, polarity, and stemness. These effects are mediated by a large number of effector molecules, which are often co-regulated by other pathways, resulting in a complex signaling network and considerable cell-type specificity. Not only with respect to effectors but also to the regulatory side, Rho GTPase signaling is complex due to the large number of activating (guanine nucleotide exchange factors (GEFs)) and inactivating (GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs)) molecules, which in turn are controlled by many different signaling pathways. This complexity explains why many aspects of Rho GTPase signaling are poorly understood, in particular with respect to the in vivo function. In this Special Issue entitled Rho GTPase Signaling in Health and Disease, we collected review articles and original papers that shed light on specific aspects of Rho GTPase function during development and disease, often with a focus on certain Rho GTPases. The role of Rho GTPase signaling in cancer has been investigated thoroughly for many years. Whereas Rho GTPases initially were simply considered invasion-promoting, pro-oncogenic molecules, recent research including cancer genome sequencing has resulted in a more differentiated view of Rho GTPase functions in cancer, revealing both promoting and inhibiting effects and high cancer-type specificity. In this Special Issue, De et al. review the role of Rac1 in solid cancers , an alteIn addition to cancer, Rho GTPase signaling has emerged as a key regulatory pathway in a number of other diseases. Guo et al. describe the role of Rho GTPase signaling in autism spectrum disorders, where 20 Rho GTPase regulator or effector genes are suggested to be disease-associated . MulherkSeveral articles in this Special Issue describe tissue-specific roles of Rho GTPase signaling. Bros et al. analyze RhoA as a crucial regulator of innate and adaptive immunity, where it regulates the migration and activation of immune cells . Rho GTPAnother set of reviews highlights specific effectors and regulators of Rho GTPases. The Cdc42-binding protein Cdc42SE1 is identified as a negative regulator of Cdc42-dependent growth signaling with potential importance in skin cancer by Kalailingam et al. . BusteloMore than three decades after their recognition as master regulators of the actin cytoskeleton, Rho GTPases and their signaling remain an exciting and active research area with many important open questions and implications in many different diseases.


Fertility preservation for both medical and non-medical reasons are gaining increasing attention world-wide. This study aimed to evaluate the awareness, knowledge and acceptance on fertility preservation in Hong Kong Chinese.This was a cross-sectional study carried out between June 2016 to March 2017. A self-administered questionnaire was distributed by convenience sampling.n = 296/697). Among them, only 54.3% were aware of any means of fertility preservation. Tertiary educated individuals are more aware than less educated individuals . The most commonly known method is oocyte cryopreservation (94.3%). Most of the respondents (80%) were interested to know about fertility preservation, 84.1% considered fertility preservation counselling necessary and 83.3% would consider undergoing fertility preservation if a treatment has a high chance of causing infertility despite the possibility of delay in cancer treatment. Up to 93.9% agreed to set up a dedicated referral centre with government funding and 73.4% agreed that fertility preservation for medical indication should be provided as a government funded service. In terms of fertility preservation for non-medical reason, 65.5 and 70.4% agreed that fertility preservation should be offered to single men and women without partner respectively, while only 53.3 and 50% agreed that fertility preservation is acceptable for delay in childbearing for career development in female and male respectively.Forty two percent of subjects returned the questionnaire (There was a low awareness but positive attitude towards fertility preservation among Hong Kong Chinese. Acceptance towards medically indicated fertility preservation is high while a considerable number also accepts it for non-medical reasons. Educational campaigns are required to arouse awareness of fertility preservation to prevent individuals being deprived of the option due to lack of knowledge. Dedicated referral centre with established efficient patient referral pathways and financial support should be provided to improve the provision and uptake of fertility preservation service to enhance the reproductive potential and life options of women. Fertility preservation is an emerging field in reproductive medicine. The advancement of reproductive technology has made childbearing possible in patients suffering from diseases or treatments that are potentially detrimental to fertility as well as healthy individuals who want to postpone their family planning for various social reasons.The majority of fertility preservation for medical indication is due to gonadotoxic treatments for cancer and autoimmune conditions, as well as other benign conditions that may impair fertility. Cancer survival has improved globally over the past decades , 2 and mElective oocyte cryopreservation for non-medical purposes has become increasingly popular in both Western communities and developing world , 10. An Despite being one of the most developed part of the world, fertility preservation service is largely underutilized and of limited availability in Hong Kong . PreviouThis was a cross-sectional survey to evaluate the awareness, knowledge and acceptance on fertility preservation among Hong Kong Chinese public. The study was conducted between June 2016 to March 2017. Ethical approval for the study was obtained from the institutional Survey and Behavioral Research Ethics Committee (SBREC).The instrument of the study was a self-administered questionnaire with a brief introduction to explain about the survey. The questionnaire was specifically developed for this study and consisted of a total of 25 items in two parts. The first part included questions on the baseline demographics, education and religious background of the participant. The second part included questions on the awareness, attitude and knowledge on fertility preservation. Their views on the demand of this service, the factors that they would consider in making any decisions for fertility preservation were examined. Practical questions on the potential costs and the need for a dedicated fertility preservation clinic were ascertained. The questionnaire was distributed to a pilot group of 30 before it derived at its final version for this study. Participants were invited to comment on the content, clarity and length of the questionnaire before the study questionnaire was finalized.The study questionnaires were randomly distributed to any individuals accessible through the public area of Obstetrics and Gynaecology, Urology and Surgical out-patient clinics at Prince of Wales Hospital, Hong Kong between June 2016 to March 2017. Medical staffs and medical students were excluded from the study. A short oral introduction of the study was made, and subjects were made aware that the questionnaire was voluntary. If the subjects agreed to participate, the questionnaire, together with a returning envelope were given to them and they were asked to mail back the questionnaire after completion anonymously.p-value of < 0.05 were statistically significant.SPSS for Window package  was used for data entry and analysis. Demographic data was summarised with the use of means, medians and percentages. The Chi-square test (χ2 test) was used for categorical data and Student’s t test (t test) for continuous variables. Results with Six hundred and ninety-seven questionnaires were distributed within the study period. A total of 296 questionnaires were completed and returned to the investigators, giving a response rate of 42.5%. Table n = 225/289) were aware that treatment for some diseases can have an adverse effect on fertility while only 54.3% (n = 157/289) of respondents were aware of any means of fertility preservation. Among them, the most commonly known strategy was oocyte cryopreservation , followed by sperm cryopreservation  and embryo cryopreservation . Significantly fewer respondents were aware of fertility sparing surgery, radiation shielding, ovarian or testicular cryopreservation . Majority of the respondents were made aware of fertility preservation by information from mass media and news , newspaper  and less from their healthcare providers . The awareness of fertility preservation was no different among different gender, age group nor marital status, yet people who had received tertiary education are more likely to be aware of fertility preservation compared to those less educated .Two hundred and twenty-five respondents  of the respondents knew where to seek fertility preservation advice or service. A minority of respondents  were aware of the existing legislations or regulations of fertility preservation service in Hong Kong.Only 21.7% (n = 214/284 and n = 239/284). Similarly, most would consider undergoing fertility preservation if there is a moderate or high chance of infertility following treatment .Majority of respondents considered fertility preservation counselling necessary if a treatment has a moderate or high chance of causing infertility  and attempt for fertility preservation procedures  despite that this might cause treatment delay of up to 4 weeks.The single most important factor they would consider to decide whether to proceed to fertility preservation is shown by Fig. More than 90% of all respondents accepts fertility preservation service for medical indications including cancer, exposure to occupational hazards that might affect fertility and men with poor sperm quality. Whereas for non-medical reasons such as single women without partner or delaying childbearing for career development, the acceptance decreases to 50 to 70% Table .Table 2n = 266/283 and 92.0%, n = 267/290 respectively). Most respondents  agreed that fertility preservation procedures for medical indication should be provided as a government funded service but less than a quarter agreed that the service should be fully subsidized.Nearly all of the respondents agreed that setting up a dedicated centre for fertility preservation is necessary and that funding should be provided by the government for this purpose  of respondents were interested to know more about fertility preservation. Most of the respondents  considered educational materials important for information gathering and decision-making in patients undergoing fertility preservation counselling, in the form of social media or websites , symposia or talks  and educational pamphlets .Up to 80.0% (In the past, cancer treatment has been focused on improvement of survival as the utmost priority while quality of life of the survivors was often disregarded. Nevertheless, cancer patients of childbearing age considered the potential loss of fertility as painful as confronting cancer itself  and thatIn Hong Kong, the currently established fertility preservation service included sperm cryopreservation, mature oocyte cryopreservation and embryo cryopreservation. Gonadal tissue cryopreservation is still limited to research settings. Access to fertility preservation service is limited as these services are only provided in two University facilities and a few private reproductive medicine centres. No public or government funded fertility preservation service is available for all indications including cancer patients. There are no established referral pathways or designated fertility preservation centres for fertility preservation counselling and only a minority of cancer patients received information about fertility preservation service and underwent the procedure. The Council on Human Reproductive Technology of Hong Kong only provides guidelines of and regulates gamete or embryo handling and storage, while there is no local fertility preservation society to promote the awareness and monitor the usage of fertility preservation service.Our study is the first to evaluate the awareness of and attitude to fertility preservation in the Chinese community. Our findings provide information on the major barriers against fertility preservation and offered insight to strategies to remove these barriers and guide the development of fertility preservation service and research in Chinese locality.We found a low awareness of fertility preservation among the respondents with only half of them ever heard of the concept of fertility preservation and only minority of them knew where to seek the service. Surveys in the western society has showed a much higher degree of awareness with up to 90% of reproductive age women aware of means of fertility preservation –20. ThisFinancial aspect is crucial in decision making for fertility preservation . In HongOur former study targeting medical practitioners in Hong Kong revealed that more than half of the clinicians expressed the concerns of potential delays in cancer treatment for fertility preservation procedures, which impeded their discussion with patients, with up to 23% considering this as the single most significant factor when deciding on fertility preservation . SurprisNearly all respondents considered fertility preservation procedures acceptable for medical reasons but more cautious for non-medical reasons. Only around half of the respondents considered delay in family planning for career development an acceptable reason for fertility preservation in women. Similar studies in the western communities revealed a much higher awareness and acceptance to elective oocyte cryopreservation. An online cross-sectional survey conducted in Denmark and UK with 973 women reported that 89% considered elective oocyte cryopreservation acceptable and up to 19% expressed active interest in the procedure . Well-knOur study has several limitations. This study was designed as a preliminary study with a limited sample size. Participants were randomly recruited by convenience sampling at hospital outpatient clinic of a single university. The aim is to reach a large population in a limited time to expedite data collection and maximize the number of recruitments but the subjects may not be representative for the entire population. Moreover, respondents who were willing to participate in this survey may represent a self-selected group with more interest in fertility preservation which is unavoidable in this form of study. This limited the generalizability of the results.In addition, our current survey was unable to reveal the reasons behind the attitude uncovered and future studies with larger sample size are required to address this issue. Nonetheless, this study was the first to evaluate the view on fertility preservation from public’s perspective in Hong Kong Chinese population. It provides important insights to the attitudes and demand of fertility preservation in a Chinese population. This information is valuable in planning of fertility preservation service and educational campaigns to promote awareness and knowledge of fertility preservation in patients and general public in Hong Kong.Our study showed an overall positive attitude towards fertility preservation among Hong Kong Chinese and highlighted the expectation of general public in fertility preservation information and service. Acceptance to medically indicated fertility preservation is high. While more than half of the individuals also accept fertility preservation for non-medical reasons. Educational campaigns utilizing all mass media and social media are required to raise patient and public awareness of fertility preservation in Hong Kong. Dedicated referral centre needs to be set up to establish efficient patient referral pathways and financial support should be provided by the government to improve the uptake of fertility preservation service.Additional file 1. Fertility Preservation Questionnaire.


Studies of immigrant fertility differentials indicate that foreign-born women have more children than native-born women, at least for some origin groups. Yet little is known about variation in cumulative fertility differentials over the life course, including the extent to which this variation develops into completed fertility differentials. This research responds with an analysis of cumulative fertility differentials in the UK for a cohort of women born between 1942 and 1971. Findings are consistent with age-specific patterns that have been documented for immigrant groups in the UK, but underline the importance of taking a cohort perspective, which helps to distinguish between the tempo and quantum of fertility. Immigrants have significantly higher completed fertility than UK-born natives if they were born in India, Pakistan, Bangladesh, Jamaica, or Western and Central Africa, but the profile of their cumulative fertility differentials—versus the UK-born—varies considerably over the life course, especially by age at migration. For example, women from Bangladesh and Pakistan have similar levels of cumulative fertility at age 40, but very different age patterns of cumulative fertility from ages 20–40. There is a consistent pattern of relatively delayed Pakistani fertility at early ages, especially for those arriving at later ages, but the same is not true for women from Bangladesh. Overall, these results imply that researchers should beware of variation in cohort fertility over the life course—with respect to both the quantum and tempo of fertility—when analysing immigrant childbearing, in addition to variation by origin and age at arrival. Research suggests that these differentials exist in almost all high income countries, especially those in Europe, North America, and Oceania , and hence can be measured at any age  , or the comparison of cumulative fertility differentials across groups. For example, comparisons by age make it possible to establish whether cumulative differentials at early ages are sustained until childbearing is completed. Alternatively, comparisons across groups can help researchers to gain insights about the broader determinants of fertility, for example by examining how cumulative fertility differentials vary by country of birth and age at migration in order to explore the social and cultural determinants of fertility.Researchers have developed numerous hypotheses to explain the fertility behaviour of immigrants and why this differs from the fertility of the destination population Coleman . These hIn this article, we do not seek to isolate and test any particular hypotheses. The aims of this research, and its research questions, are first and foremost descriptive. Nevertheless, a comparison of life course differentials can help to narrow the potential list of explanations for the fertility of a given migrant group. It can also show which groups, and which stages of the life course, merit further investigation. As such, although it may not be possible to carry out a robust test of specific hypotheses without bespoke research designs, the analysis of life course differentials can provide an indication that some hypotheses are more plausible than others. This is particularly the case, when the analysis disaggregates migrants by origin (e.g. country of birth) and age at migration. It is for this reason that this section describes the hypotheses that are used to explain immigrant fertility   and that fertility is elevated after migration because migration is linked to partnership behaviour (family formation) . Convergence over exposure to destination can therefore be evaluated by comparing how life course differentials vary by age at migration. Similar to research on ethnic fertility differentials, this analysis can be used to investigate exposure to destination as a determinant of fertility.Adaptation predicts immigrant fertility convergence over the life course  due to exposure to destination norms and institutions, or due to adaptation to new socio-economic circumstances (Andersson and Scott elevated fertility), as long as these profiles then gradually disappear with age . However, adaptation is hard to assess for adult migrants because elevated fertility after migration might have a range of alternative explanations  or that women’s propensity to migrate is increased if they do not have a child  , there have been many researchers who have used a cohort approach to study fertility , or on the examination of part of the reproductive life course, (e.g. first birth rates). There has been limited prior analysis of the profile of differentials across the reproductive life course of immigrants, and no previous research that allows us to distinguish between tempo and quantum variation within and between immigrant origin groups. This is an important gap because, unlike other approaches, such an analysis makes it possible to understand distinctions between the tempo and quantum of immigrant fertility. This study therefore aims to redress this gap by describing how immigrant fertility differentials vary over the reproductive life course, and how this life course variation is different for different groups of immigrants. In order to do this, it carries out an empirical study of the UK.There are several reasons why the UK is an excellent case for the study of immigrant fertility, especially in Europe. Compared to most other high income countries, the UK has a long history of immigration from a diverse range of origins , which are representative of the UK population, and includes responses for more than 50,000 adults who were surveyed between 2009 and 2011  gave slightly higher estimates of average fertility  number of children born to immigrants is compared with the number of children born to natives at the same age. This comparison is presented as a ratio, which represents the immigrant fertility differential for a given group of immigrants at a given age.It is important to note that all births up to age 40, before and after migration, are known for all women in the sample, so for the purposes of this analysis fertility is complete. Given that births are a rare event over the entire life course, comparisons are made by single years of age. This is important because comparisons of differentials between groups are likely to be highly sensitive to even small changes in childbearing, especially since the average completed fertility of both immigrants and natives is not much more than two children per woman.Comparing the unweighted counts of foreign-born and UK-born women in the analytical sample, they appear to have similar distributions across several covariates that are relevant for the study of fertility, including education and partnership. However, covariates are not used in this analysis, not least because most relevant covariates (like partnership and education) are simultaneous to the fertility process , and their inclusion (given our research questions) would therefore bias the findings and make them very difficult to interpret.The statistical analysis uses count regression models to estimate children ever born at each age Agresti . These mi at age j. As the only explanatory variable, i = 1,…, n, estimated separately for each age j = 20,…, 40, where j, a risk ratio for each migrant group, compared to the reference group of UK-born women, is therefore defined as: The models are defined as follows: Let svy command in Stata version 11, to account for the complex survey design of the UKHLS . For example, the IRRs for Indians in Fig. Figure Based on a comparison of origins at age 40, we can identify the origin groups that have the largest completed fertility differentials. These immigrants—Jamaicans and South Asians —therefore make the largest eventual contribution to population size in terms of their fertility rates . However, it is also apparent that they have very different profiles, such that their fertility impacts population dynamics in different ways at different ages. For example, compared with Pakistani women, Bangladeshi women have very similar completed fertility, but far higher differentials at ages under 30. The quantum of fertility at age 20 is almost the same as natives for Pakistani-born women, whereas births to Bangladeshi-born women are (on average) much earlier than both Pakistanis and natives. A similar comparison can be made between women from India and Jamaica, who have very similar completed fertility, but very different profiles, such that the childbearing of Jamaicans begins much earlier . Overall, these patterns suggest that while Bangladeshis and Pakistanis may eventually make the greatest relative contribution to population size (i.e. net of their absolute numbers), Bangladeshis and Jamaicans (in these cohorts) were more likely to have an earlier (relative) impact on population change via their (early) childbearing.For some origin groups, the profile of differentials is more stable over the life course. This is the case for African and Middle Eastern origins, as well as women who were born in the Caribbean outside Jamaica. However, even among these aggregated groups, there is evidence of differences in birth timing. Differences in timing are also evident for origin groups that have lower levels of completed fertility to natives . For example, Southern and Eastern European migrants are much less likely to have children at young ages, as compared with UK natives. At age 20, their average number of children born is lower than UK-born women by a factor of 0.08 . This compares with a factor of 0.85 at age 40 , which although still below 1.0, is much more similar to the native norm.For Southern and Eastern European migrants, this pattern may reflect the (relatively lower) fertility of their origin countries, which is not only the case for recent estimates of period fertility, but also for cohort fertility for the cohorts that are studied here , which suggests that this pattern of childbearing is not necessarily associated with origin fertility norms. Alternative explanations, which could be true for all origin groups that exhibit negative differentials, is that this pattern of differentials may be driven by disruption, or by the selection of migrants who are more likely to postpone childbearing and end their reproductive lives with fewer children than UK-born natives.As discussed, age at migration is likely to be linked to changing patterns of immigrant fertility differentials. The majority of immigrants arrive as adults, which means that they migrate after the start of their reproductive years. For example, more than two-thirds of immigrants who were born outside the UK and resident in England and Wales in 2011 had an age at arrival between 15 and 44 ONS .One of the difficulties for assessing fertility differentials for adult migrants is the fact that the timing of their childbearing and their immigration are likely to be associated with each other . A similar pattern is observed for Pakistanis, and to a lesser extent Indians. It is likely that these patterns of lower pre-migration differentials are due to a combination of the postponement of childbearing until after migration (e.g. for women who intend to migrate), alongside the lower likelihood that women will migrate if they already have children . However, this pattern is not universal and cannot be used to explain the age profile of differentials for Bangladeshi women who migrate after age 25.These patterns of lower pre-migration differentials are not surprising given previous research, and when combined with evidence of higher differentials after migration, they go some way towards explaining the established finding of elevated fertility soon after migration for adult migrants  common trends. This has implications for future research, including whether the period TFR is likely to be distorted by tempo-variation, in particular by elevated fertility after arrival for adult immigrants. It is well known that comparisons between groups using the period TFR can be distorted by differences in their timing of births . Moreover, this suggests that if adaptation does occur, then it is more likely to occur towards the end of childbearing, presumably because there is less time to adapt at the beginning of the life course.By contrast, Pakistani child migrants show a very different pattern from these origins. The fact that they have almost no differential at early ages, but that their differential steadily increases with age, suggests that their relative contribution to population growth (net of their absolute numbers in the population) will be very different from other child migrant groups. This also implies that there is a different explanation for their differentials. As outlined in the introduction, most hypotheses, except cultural entrenchment, predict that the fertility of child migrants should be the same as, or converge towards native fertility . Of all the African groups, only West and Central Africa demonstrates a lot of variation by age at migration, with the most distinct pattern being for those arriving aged 26–35.For the remaining origin groups, who all have lower completed fertility than natives on average, there are many similarities in the patterns of differentials by age at migration. Irrespective of age at migration, adult migrants in these groups generally display significantly lower numbers of children born at early childbearing ages, although this difference becomes smaller with age. This can be contrasted with the profile of differentials for child migrants from these groups, which are much closer to the average for natives. The exception is migrants from South and East Europe, where child migrants exhibit the same profile as adult migrants. These results suggest that the timing of migration for South and East European immigrants makes very little difference to their fertility. As noted in previous research, studies of migrant fertility often ignore the fertility of immigrants from origins that have lower fertility than the destination  are higher than estimates of completed fertility . Our results also demonstrate, using longitudinal data for completed fertility profiles, that this is likely to be due to elevated fertility after immigration, (at least for adult immigrants from a large number of immigrant origin groups). These findings are new, although the existence of elevated fertility after arrival has also been proposed by prior research comparing the ASFRs of immigrants and natives in the UK before and after immigration Dubuc , 2016, oAs in prior research, it is important to be cautious in interpreting our evidence of elevated fertility after arrival. In particular, it is important not to compare fertility before and after migration in order to draw conclusions about the  association between migration and fertility , all have quite similar patterns of differentials to each other. This suggests that it may be reasonable to group them together, and that research on these origins might best be directed towards the early childbearing ages.Of course, there are limitations to these findings, and the extent to which they can be generalised remains uncertain. It is important to note that these results are for particular cohorts of women who have completed their fertility, and the childbearing of women from later birth cohorts may well be different. Future research could extend the approach taken here and investigate cumulative fertility differentials for cohorts of women who have not yet completed their childbearing. It is also important to consider that this issue of generalisability not only applies to immigrants but also to natives. These results are with reference to UK-born women, who may themselves have particular fertility patterns, such as higher levels of teenage childbearing, as compared with women in other immigrant destinations. Another limitation is that these results are based on those immigrants who remain in the UK, thereby excluding those who emigrate or die between arrival and time of survey. Added to this are some specifics of the sample, such as the exclusion of immigrants arriving aged 36 and over that may further affect the representativeness of these results.Despite these limitations, and the need to be cautious when interpreting the findings, these results for the UK also provide indicative evidence for and against some prominent hypotheses that have been used to explain migrant fertility behaviour. For example, the existence of positive differentials across the life course for child migrants from Pakistan and Bangladesh, including with respect to their completed fertility, suggests that their fertility preferences may be culturally entrenched. As noted, this result might be due to socio-economic ‘entrenchment’, rather than the influence of ‘culture’. Nevertheless, in contrast to most other origin groups, this is tentative evidence against childhood socialisation , and suggests that these groups may be worthwhile to study in future research. However, it is also important to note that, on its own, the absence of fertility differentials for child migrants is not enough to demonstrate childhood socialisation. A more reliable test of childhood socialisation would require data that more closely examines the interaction between migration background and childhood context.Regardless of the explanation for these patterns, these results highlight the fact that it may be inappropriate to make generalisations about the quantum and tempo of immigrant fertility, as compared with natives. Conclusions about immigrant fertility differentials are very likely to depend upon the way that fertility is measured and the groups that are investigated. Similarly, these results show that the composition of the immigrant population will be very important in determining immigrant fertility differentials, and this includes the composition of the samples that are analysed. For example, the analysis of samples that include women who have not yet finished their childbearing may have a material impact on any conclusions about the magnitude of differentials, both now and in the future.


Background. Human T-cell lymphotropic virus type 1 (HTLV-1) is responsible for tropical spastic paraparesis and HTLV-1-associated leukemia/lymphoma. The infection is endemic in some areas of Peru, but its prevalence in the Peruvian Amazon is not well established. We aimed to assess the seroprevalence of HTLV-1 infection in pregnant women in the Peruvian Amazon. Moreover, we performed a systematic literature review and meta-analysis of the seroprevalence of HTLV infection in Peru. (2) Methods. This is a prospective cross-sectional study involving pregnant women attending health centers in the city of Iquitos, Peru, in May and June 2019. The presence of antibodies against HTLV-1 was assessed using ELISA . Positive cases were confirmed by Western Blot and HTLV-1 proviral load. (3) Results. The study included 300 pregnant women with a mean age of 26 years (standard deviation [SD] 6.4). Five patients were diagnosed with HTLV-1 infection  0.7% to 3.8%). Pregnant women with HTLV-1 infection were discretely younger . None of the five women had been transfused, and all were asymptomatic. Two (40%) also had a positive serology for Strongyloides, but larvae were not detected in any of the parasitological stool studies. The systematic review component identified 40 studies, which showed that the prevalence of HTLV infection in the general population was 2.9% (95% CI 1.2% to 5.3%) and in women of childbearing age, 2.5% (95% CI 1.2% to 4.0%). (4) Conclusion. The prevalence of HTLV-1 in the Peruvian Amazon basin is about 1.7%, indicating an endemic presence. Screening for HTLV-1 in prenatal care is warranted. Human T-cell lymphotropic virus (HTLV) was the first human retrovirus identified. It is mostly sexually transmitted  but it cHTLV can spread through mother-to-child transmission (MTCT), mainly through breastfeeding. As this is one of the main mechanisms of transmission in endemic areas, systematic HTLV antenatal screening is carried out in some countries, such as Japan and a few Latin American countries like Brazil ,7. MotheWorldwide, approximately 5 to 10 million people are infected with the human T-cell lymphotropic virus type 1 (HTLV-1) ,9. A larKnowledge on the prevalence of HTLV-1 is vital for understanding whether antenatal screening is justified in the Amazon Basin, so that infected mothers can avoid breastfeeding to effectively prevent vertical HTLV transmission. Thus, this study aimed to assess the seroprevalence of HTLV-1 infection in pregnant women in the Peruvian Amazon. In addition, we undertook a systematic literature review and comprehensive review of the incidence of HTLV-1 in Peruvian cohorts and meta-analyzed the seroprevalence of HTLV infection in Peru.We included 300 pregnant women with an average age of 26 years (standard deviation [SD] 6.4 years). The mean number of deliveries was 2.9 (SD 1.7), and the mean gestation period, 172 days (SD 59). Just under half (44.7%) lived in peri-urban areas, while the rest lived in the city.p = 0.13). None of the five women with HTLV-1 had been transfused, had a tattoo, or had a history of leukemia or neoplasm. All of them with other children had breastfed in the past. Strongyloides serology was positive in 2 of the 5 pregnant women (40%); however, the parasitological study in stool was negative for Strongyloides larvae in all of them. All HTLV-infected women were asymptomatic.Five patients were diagnosed with HTLV-1 infection by serology . Pregnant women with HTLV-1 infection were slightly younger . The pooled proportion of HTLV in the general population was 2.34%  [Strongyloides in HTLV-infected women in our study. The Peruvian Amazon may be an endemic region for Strongyloides, signaling high prevalence in the general population, including pregnant women [Strongyloides infection in pregnant, HTLV-infected women.Incidence of patients . HTLV inividuals . Moreovenfection . In our ultures) ,61. Thernt women . Future The main strength of this study is its evaluation of HTLV-1 prevalence in pregnant women in greater Iquitos, providing information that is relevant for monitoring the risk of perinatal transmission through breastfeeding. This study has several limitations, Firstly, it screened a small sample for a prevalence study, so differences in patient characteristics between positive and negative cases could not be detected. Secondly, the proviral load was determined retrospectively, and data could not be collected from one patient who was not followed up. Thirdly, it was not possible to follow up the pregnant women and recommend avoiding breastfeeding for preventing transmission.Strongyloides, to provide definitive evidence supporting implementation of screening in pregnant women, as already in place for HIV and other viral infections.We consider necessary a large study in the Peruvian Amazon to understand the relevance of asymptomatic HTLV infections in pregnant women and the coinfection with We performed a cross-sectional survey of HTLV-1 and strongyloidiasis in May and June 2019.We included pregnant adults (≥18 years) attending health centers in four districts of greater Iquitos, a city in the Peruvian Amazon. Participants were selected through convenience sampling  when they visited the midwife for prenatal check-ups. Previous studies performed by our group have reported details of the study population and data collection ,62.6 PBMCs by a spin column using the QIAamp blood DNA mini kit .The presence of antibodies against HTLV-1 was assessed by ELISA . Positive cases were confirmed by Western blot and SYBR Green–based real-time quantitative polymerase chain reaction (qPCR) assay, developed in the Institute of Tropical Medicine ‘Alexander von Humboldt’, Peruvian University Cayetano Heredia, Lima . BrieflyStrongyloides stercoralis with the Strongyloides IgG IVD-ELISA kit , while Baermann’s method and charcoal culture were performed in stool specimens from HTLV-1 positive patients.Serology was processed for p value was less than 0.05.Patient information was recorded in a Microsoft Excel database. The collected data were analyzed using SPSS statistical software . We used the chi-squared test or Fisher’s exact test to analyze the presence of HTLV infection according to several demographic variables, considering results to be significant when the The Ethics Committee of the General University Hospital of Alicante (Spain) approved the project (PI2018/113), as did the Ethics Committee of Loreto Regional Hospital in Iquitos (Peru) (027-CIEI-HRL-2019). After receiving information on the study, individuals who volunteered to participate gave their written consent and were included. All results were kept strictly confidential, and participants with positive results were referred to their health centers for assistance or treatment.We performed an electronic search in PubMed and SciELO on 1 April 2020, using the following key words, grouped into three main concepts: “HTLV” AND “Peru”. Studies collected were published from 1 January 1991 to 1 April 2020. The search was limited to studies in humans and written in English, Spanish, Italian, or French. Additional records were identified by means of cited reference searching and electronic searches for gray literature (Google and Google Scholar).HTLV were extracted, regardless of the type of population .We examined surveys, reports, reviews, and epidemiological studies on the prevalence of HTLV. Titles and abstracts were screened for relevant papers, and full texts were retrieved when appropriate. Data on the prevalence of 2 statistic  [Strongyloides infections, (8) people with tuberculosis, and (9) blood donors. We use a random-effects (inverse variance) method.We performed a proportion meta-analysis, using the Stuart-Ord (inverse double arcsine square root) method to calculate the 95% coefficient intervals and create the forest plots. Heterogeneity was analyzed using the I. 3.3.4) . The preThe prevalence of HTLV-1 in the Peruvian Amazon basin is about 1.7%. Since HTLV is endemic  in this population of pregnant women, screening for HTLV in prenatal care is warranted.


Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens, respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1–45), IMD (aa 46–67), CIL (aa 68–93), TMD (aa 94–119), and CTD (aa 120–131). In vitro, the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections.Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper ( Interferon-stimulated genes (ISGs) are induced by interferon (IFN) through a series of signal transduction cascades, and exert virous antiviral effects at specific stages of the virus life cycle  –3. IFN-iEpinephelus spp.), a commercial cultured fish species in China and Southeast Asian countries (Epinephelus tauvina), belonging to the genus Ranavirus, family Iridoviridae  and RNA2 (1.4 kb) (Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) are important viral pathogens of groupers  . To clar(1.4 kb) , 33, num(1.4 kb) , IRF7 (3(1.4 kb) , mitocho(1.4 kb) , stimula(1.4 kb) , and cho(1.4 kb) . Recentl(1.4 kb) . Whetherin vitro. Our findings provided new evidence that EcIFITM1 played a bi-functional role in fish virus infection, including immune regulation and lipid metabolism.In this study, an IFITM1 homolog from orange spotted grouper (EcIFITM1) was cloned and characterized. The subcellular localization of EcIFITM1 was observed and its antiviral roles against both fish DNA and RNA viruses were investigated. In addition, the effects of EcIFITM1 on lipid metabolism and the host interferon response were determined E. akaara, respectively, were cultured at 28°C in Leibovitz's L15 medium  containing with 10% fetal bovine serum  (The grouper spleen (GS) and grouper brain (GB) cells which derived from the spleen and brain tissues of co, USA) . SGIV anco, USA) , 41. Virhttp://www.ncbi.nlm.nih.gov/blast), we carried out the sequence analysis of EcIFITM1. In addition, the conserved domains were predicted using InterPro (http://www.ebi.ac.uk/interpro). Multiple amino acid (aa) sequence alignments were created using ClustalX1.83 software and edited with GeneDoc. A Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0 software.Based on the expressed sequence tag (EST) sequences of EcIFITM1 from the grouper spleen transcriptome , the opein vitro, the relative expression of EcIFITM1 was detected by quantitative real-time PCR (qPCR) after SGIV or RGNNV infection, respectively. In brief, GS cells were infected with SGIV or RGNNV at a multiplicity of infection (MOI) of 2. The infected cells were harvested at 3, 6, 18, 24, and 30 h post-infection (h.p.i.) for qPCR analysis.To elucidate the changes in EcIFITM1 expression in response to fish virus infection in vitro, EcIFITM1 was subcloned into pEGFP-C1 and pcDNA3.1-3 × HA using the primers listed in To elucidate the potential function of EcIFITM1 To determine the subcellular localization of EcIFITM1, GS cells were seeded into glass bottom cell culture dishes (35 mm), and co-transfected with 0.5 μg pEGFP-C1 or pEGFP-EcIFITM1 with 0.5 μg pDsRed2-ER , pDsRed250). In addition, the level of RGNNV-CP protein in the pcDNA3.1-3 × HA- or HA-EcIFITM1-overexpressing cells were evaluated by an indirect immunofluorescence assay as described below.To evaluate the effects of EcIFITM1 overexpression on viral replication, GS cells were transfected with pcDNA3.1-3 × HA or HA-EcIFITM1 for 24 h, followed by SGIV or RGNNV infection (MOI = 2), respectively. First, the cellular morphology was observed and photographed using a phase contrast microscope. Next, the virus-infected cells were harvested at 12 h.p.i. and 24 h.p.i. for qPCR and western blot. The SGIV-infected cells were collected at 24 h.p.i. and the viral titers were determined on GS cell monolayers by a 50% tissue culture infective dose  oligonucleotides targeting different sequences of EcIFITM1 were designed and commercially synthesized by Invitrogen . GS cellTo determine the detailed roles of EcIFITM1 at the early stage of RGNNV or SGIV infection, qPCR assay and single-particle tracking technique were used to analyze the viral entry into the host cells. In one group, the GS cells were seeded into 24-well plates and transfected with HA-EcIFITM1 or EcIFITM1-specific siRNA for 24 h. The transfected cells were then infected with SGIV or RGNNV at 4°C to allow for synchronous virus adsorption into the cells. After 1 h, the GS cells were washed three times with cold serum-free medium to remove any unbound virus, after which a pre-warmed medium was added to the culture for another 4 h (SGIV) or 1 h (RGNNV) at 28°C. Non-internalized viruses were removed by washing the cells with citrate buffer  for 1 min , 38. TheIn another experimental group, GS cells were grown in glass bottom cell culture dishes (35 mm) and transfected with pEGFP-EcIFITM1 or pEGFP-C1 for 24 h. The cells were pre-chilled at 4°C for 5 min and infected with purified SGIV particles labeled with Alex-Fluor 647 labeled SGIV (MOI = 10) as previously described , 44, 45.−ΔΔCT method. The data were represented as the mean ± standard deviation (SD).Total RNA was extracted using an SV Total RNA Isolation Kit  and reversed with a ReverTra Ace qPCR RT Kit  according to the manufacturer's protocol as described previously . qPCR waThe total cell proteins were extracted with Pierce RIPA Lysis buffer , and the protein concentration was measured using a BCA protein assay kit  according to the manufacturer's instructions. The extracted proteins were electrophoresed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes  for 60 min, and blocked with 5% (w/v) skim milk in Tris-buffered saline  containing 0.5% Tween 20. The separated proteins were incubated with the following specific primary antibodies: anti-HA , anti-SGIV major capsid protein (MCP)  (prepared in our lab), anti-RGNNV capsid protein (CP)  (prepared in our lab), and anti-β-actin  for 2 h at room temperature. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated sheep-rabbit IgG or sheep-mouse IgG at a dilution of 1:5,000  for 2 h at room temperature. Immunoblots were visualized using an enhanced HRP-DAB Substrate Chromogenic Kit  according to the manufacturer's protocol. The intensity of each protein was quantified using Image J software and normalized to the expression of β-actin.GS cells transfected with pcDNA3.1-3 × HA or HA-EcIFITM1 were infected with RGNNV (MOI = 2) for IFA as previously described . Briefly6 cells/well; n = 6) were collected, and were centrifuged at 4°C, 300 × g for 10 min. After washing once with cold PBS, the cells were centrifuged at 4°C, 300 × g for 10 min. One well of the samples was collected for western blot analysis as described above and the rest were immediately frozen on ice and stored at −80°C until further use.Sample preparation: GS cells were grown in six-well cell culture plates at 28°C for 18 h, and transfected with pcDNA3.1-3 × HA or HA-EcIFITM1 for 36 h. The transfected GS cells , vortexed for 30 min, and centrifuged to separate the aqueous phase from the organic phase for further metabolomics analyses.2O, MeOH, and ACN at a ratio of 1:1:1 with 5 mM NH4Ac. Phase B consisted of IPA and CAN at a ratio of 1:1 with 5 mM NH4Ac. Gradient conditions were as follows: 0.5 min, 20% phase B; 1.5 min, 40% phase B; 3 min, 60% phase B; 13 min, 98% phase B; 13.1 min, 20% phase B; 17 min, 20% phase B. An MS system was AB Sciex TripleTOF® 6600, using electrospray ionization (ESI) under the positive ion modes. The mass scanning range was 100–1,200 m/z and the mass parameters were as follows: curtain gas, 35.00 psi; ion source gas 1, 50.00; ion source gas 2, 50.00; ionspray voltage, 5,500.00 V; and temperature, 600°C.Ultra-performance liquid chromatography (UPLC)-QTOF-Mass spectrometry (MS) profiling analysis: A UPLC system used was Shimadzu UPLC lc-30a with Phenomenex Kinete C18 column . Injection volume, 1 μL; flow rate, 0.4 mL/min; column temperature, 60°C; sample chamber temperature, 4°C. Phase A consisted of Ht-test.Data preprocessing and statistical analysis: The data was edited into a two-dimensional data matrix using Excel 2010 software, including the retention time (RT), mass to load ratio (M/Z), molecular formula of substance, and compounds. Principal components analysis (PCA) and orthogon partial least squares-discriminant analysis (OPLS-DA) were used to test the metabolic differences between the control and treatment groups. Differential metabolites were screened based on the variable importance in projection (VIP) (≥ 1) from an OPLS-DA model and absolute Log2-fold change (FC) (≥1) obtained from a two-tailed Student's Metabolite extraction, UPLC-QTOF-MS, data preprocessing, and statistical analysis were completed using Wuhan MetWare Biotechnology Co., Ltd.p-value was < 0.05 (*p < 0.05).The statistical analyses were performed using SPSS version 20. A one-way ANOVA was used to evaluate the differences between groups. Differences were considered statistically significant when the MW118611). The homology analysis results indicated that EcIFITM1 shared 64 and 43% identity with the IFITM1 homolog from greater amberjack, S. dumerili (XP_022611970.1), and humans, Homo sapiens (NP_003632.3), respectively. The amino acid alignment analysis indicated that EcIFITM1 contained five domains: (1) a variable hydrophobic N-terminal domain (NTD) (aa 1–45); (2) a conserved hydrophobic intramembrane domain (IMD) (aa 46–67); (3) a conserved intracellular loop (CIL) (aa 68–93); (4) a variable hydrophobic transmembrane domain (TMD) (aa 94–119); and (5) a highly variable C-terminal domain (CTD) (aa 120–131)  and 346 bp of the 3′-UTR with a poly (A) tail (GenBank Accession No: 120–131) . The phyn IFITMs .To analyze the expression pattern of EcIFITM1 in response to different viruses, GS cells were infected with RGNNV or SGIV, and collected at the indicated time points for qPCR. As shown in in vitro, pEGFP-C1 or pEGFP-EcIFITM1 were co-transfected with pDsRed2-ER, pDsRed2-Mito, pDsRed2-Rab5, or pDsRed2-Rab7 into GS cells. Moreover, at 48 h post-transfection, GS cells transfected with pEGFP-C1 or pEGFP-EcIFITM1 were stained with Lyso-Tracker. As shown in Several literatures demonstrate that IFITMs are localized in the early and late endosomes , lysosom50, qPCR, western blot, and IFA, respectively. First, the level of mRNA and protein expression of EcIFITM1 in HA-EcIFITM1- or pcDNA3.1-3 × HA-overexpressing cells was evaluated. The results showed that HA-EcIFITM1 was successfully overexpressed in transfected cells  and ceramide synthase (Cers) played an antiviral role in IAV infection  was cloned and its role in fish virus infection were investigated in this study. EcIFITM1 encoded a cytoplasmic protein, and partly colocalized with EEs, LEs, and LYs. Moreover, the ectopic expression or knockdown of EcIFITM1 The datasets generated in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/YZ completed the main experiments, analyzed the data, and wrote the manuscript. LW, JZ, LH, SW, and XH participated in the preparation of reagents and virus stocks, and confocal microscopy analysis. QQ and YH conceived and supervised the study, and edited and reviewed drafts of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although considerable research highlights the interactions between obstructive sleep apnea-hypopnea syndrome (OSAHS) and cardiovascular diseases, the effect of mandibular advancement device (MAD) treatment on cardiovascular complications in OSAHS patients remains unclear. We evaluated the effect of OSAHS treatment with MADs on the myocardium. All methods in this study were in accordance with relevant guidelines and regulations of the medical ethics committee in Hospital of Stomatology, Hebei Medical University approved the work. Thirty New Zealand rabbits were randomized into three groups: the control group, Group OSAHS, and Group MAD. Hydrophilic polyacrylamide gel was injected into the soft palate of the rabbits to induce OSAHS. In Group MAD, a MAD was positioned after OSAHS induction. All animals were induced to sleep in a supine position for 4–6 h/day for 8 weeks. Echocardiography was used to determine the structure and function of the heart. The histological changes were detected by optical microscopy and transmission electron microscopy (TEM). The levels of ET-1(endothelin-1) and Ang II (Angiotensin II) in the plasma were measured by an enzyme-linked immunosorbent assay (ELISA). The expression of ET-1 mRNA in heart tissue was detected by RT-PCR. Histological abnormalities, left ventricular hypertrophy, and left ventricular dysfunctions were demonstrated in Group OSAHS, and the abnormities were rescued with MAD treatment. Higher levels of plasma ET-1 and Ang II and elevated expression of ET-1 mRNA in cardiac tissue were detected in Group OSAHS compared with Group MAD and the control group. The blood oxygen saturation was negatively correlated with the levels of ET-1 and Ang II. OSAHS-induced elevated levels of ET-1 and Ang II may be attributed to myocardial structural abnormalities and dysfunction. Early treatment of MADs may play an important role in preventing myocardial damage in OSAHS rabbit model. It is characterized by recurrent partial or complete closure of the upper airway during sleep, which leads to oxygen desaturation and arousal from sleep. OSAHS is a potential major risk factor for the morbidity and mortality of many cardiovascular complications2, such as hypertension, arrhythmias, myocardial infarction, heart failure, ventricular hypertrophy, systolic and diastolic dysfunctions, as well as stroke4. OSAHS is associated with a significantly higher risk of hypertension and is regarded as an independent risk factor for hypertension5. Evidence indicated that the cardiac dysfunction and cardiomyocyte injury was detected in OSAHS patients and animal modes7. The cardiac dysfunction might be associated with a decreased coronary tone, and an intact coronary endothelium during OSAHS8. Previous studies suggested that OSAHS was contributed to microvascular endothelial dysfunction and augmented vasoconstriction9, which was associated with endothelial system. The renin-angiotensin system plays critical roles in maintaining normal cardiovascular functions and regulates extracellular fluid volume and blood pressure. This system may be potentiated by increased sympathetic system activity, and increased levels of insulin and leptin that are frequently present in OSAHS. Sympathetic nervous system overactivity could result in the overproduction of ET-1(endothelin-1) and Ang II (Angiotensin II), which are important to vascular tone and blood pressure. ET-1 and Ang II are mainly produced by endothelium and play an important role in cardiovascular alterations. Ang II induces several forms of cardiac dysfunction including hypertrophy, arrhythmia, and ventricle function failure11. Sleep apnea patients have been shown to have higher angiotensin II concentrations compared to healthy subjects13. Therefore, we speculated that OSAHS induced cardiac dysfunction and abnormal structure, which might be associated with ET-1 and Ang II levels.Obstructive sleep apnea-hypoxia syndrome (OSAHS) affects 9–38% of the general adult population, 13–33% of men and 6–19% of women, and prevalence rates increase with age14, continuous positive airway pressure (CPAP) treatment16, mandibular advancement devices (MADs)18, and surgeries to enlarge the upper airway, such as uvulopalatopharyngoplasty (UPPP) and maxilla-mandibular advancement osteotomy19. It is popular to use MADs for most OSAHS patients, especially those unable to tolerate CPAP therapy, due to their many advantages, such as being traumatic, convenient, and low cost. It has been shown that OSAHS treatment might reduce the risk of cardiovascular complications. CPAP treatment has been proven to reduce blood pressure20 and correct left ventricular hypertrophy and systolic and diastolic dysfunctions caused by OSAHS22. There were limited data on the effects of oral appliance treatment on cardiac function. Both Hoekema et al.23 and Barnes et al.24 used echocardiography to image left ventricular structures and function and did not find any effect of oral appliance on this heart function parameter after 3 month of treatment. A recent meta-analysis showed the other studies on MADs treatment on the cardiovascular system associated with OSAHS were limited to the changes in blood pressure, heart rate and fatal cardiovascular events25. Therefore, there is a lack of good quality data assessing the effect of MADs on cardiovascular system.OSAHS therapies include behavioral treatment27 and to determine the effects of OSAHS on cardiac structure and function and the role of MADs in the remedies of possible injuries. This will provide evidence for the clinical treatment of OSAHS with MADs and further explore the possible mechanism of these changes.In the present study, we aimed to investigate the changes of cardiac structure and function in the rabbit OSAHS model developed in our laboratoryAll methods in this study were performed in accordance with the medical ethics committee in Hospital of Stomatology, Hebei Medical University. Animal use and care was in accordance with the guidelines of the medical ethics committee for the housing and care of animals bred, supplied and used for scientific purposes. All experiments were performed in accordance with relevant guidelines and regulations. The medical ethics committee in Hospital of Stomatology, Hebei Medical University approved the work (Certificate No. [2017]013).26. In brief, the animals of Group OSAHS and Group MAD were induced to develop OSAHS by gel injection into the center of soft palate, approximately 1.5 cm away from the junction of the hard and soft palates. The MADs were given to the OSAHS rabbits in Group MAD on day 3 after OSAHS development. Animals in the control group were not given any treatment.Thirty 6-month-old male New Zealand white rabbits (body weight 3.0–3.5 kg) were randomly divided into three groups, the control group, Group OSAHS, and Group MAD, with 10 animals in each group. The OSAHS rabbits were induced as previously described in our studyThe animals of all three groups were fixed in a homemade device to simulate the supine position. A CBCT scan of the upper airway was performed using a CBCT machine. The scan parameters were set to 120 KV of voltage, 5 mA of current, 0.3 mm of layer thickness, and 17.8 s of scan time. A single 360° rotation scan was performed from the cranium to the clavicle. Then, the retropalatal space was examined by measuring the sagittal diameter of the upper airway from the top to the 1/4, 1/2, and 3/4 level of the soft palate.27. Hypopnea was defined as a reduction between 20% and 50% in nasal flow for at least two breaths. Apnea was defined as an absence of airflow at the nose and mouth for longer than two breaths28. The apnea-hypopnea index (AHI), the number of apneas and hypopneas per hour during sleep throughout the whole night, was scored for all animals.PSG  was used to monitor the sleep parameters. PSG recordings were conducted as described in our previous studies26. Briefly, MADs were made of self-curing composite resin with a 30° inclined plane to the long axis of the upper incisors and was bonded to the upper incisors with glass ionomers. The mandible was guided forward approximately 3–4 mm. CT and PSG were used to evaluate the effectiveness of MAD treatment following the protocols described above.The fabrication of MADs was performed according to the previous protocol developed in our laboratoryAll animals were induced to sleep in a supine position for 4–6 hours per day for eight consecutive weeks. After eight weeks, heart tissues were harvested and embedded in paraffin after fixation in 10% buffered formalin. Sections were stained with hematoxylin/eosin and examined under light microscopy . A portion of the cardiac apex was quickly dissected and fixed in 4% cryopreservation glutaraldehyde solution and then viewed by transmission electron microscopy.Echocardiographic analysis was performed by a high-resolution ultrasound imaging system  with an IE33 transducer. First, the rabbits were held in the supine position, the anterior chest wall was shaved, warm ultrasound gel applied to the chest, and the transducer probe was placed over the left hemithorax. Multiple short axis M-mode images of the left ventricle were obtained, and these images were analyzed for left ventricular (LV) functional parameters in triplicate. The other indices measured included the LV wall thicknesses [LV posterior wall (LVPW) and interventricular septum (IVS)], right ventricle diameter (RVD), and LV chamber dimensions (LVID) as recommended by the American Society of Echocardiography. M-mode recordings detected the left ventricular end-diastolic diameter (LVEDd) and left ventricular endsystolic diameter (LVEDs). The left ventricular endsystolic volume LVESV = 7/(2.4 + LVEDs) × LVEDs3 × 1000, left ventricular end-diastolic volume LVEDV = 7/(2.4 + LVEDd) × LVEDd3 × 1000, and left ventricular ejection fraction (LVEF) = (LVEDV − LVESV)/LVEDV × 100%. Four-chamber echocardiography showed the maximum flow rate in the early diastole (E), maximum flow rate in the systolic phase (A) of the mitral valve.Two milliliters of blood from the external jugular vein was sampled and immediately centrifuged at 3000 rpm for 10 minutes. The plasma was collected to measure the level of ET-1 and Ang II by an ELISA kit .To examine the correlations between ET-1 and Ang II in hypoxic plasma, a regression analysis was conducted between the level of ET-1 and Ang II with blood oxygen saturation.Total RNA was extracted from the heart using Trizol reagent  and was reverse transcribed by oligo(dT) priming, and first-strand cDNA was synthesized . The resultant cDNA was amplified using Taq DNA polymerase, and the fluorescent signal generated was analyzed automatically by ABI 7500 v 2.0.1 software. Relative quantification was used to evaluate the gene expression of the samples.ET-1, Forward: 5′-GACCACAATGACTTTGGCGTAT-3′;Primer sequence: Reverse: 5′-CGATGGCTTCAGGGATGG-3′;GAPDH, Forward: 5′-ACCTGACCTGCCGTCTAGAA-3′;Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′SPSS 22.0 software  was used for data analysis. Different parameters were determined for normally distributed data. All data were expressed as the mean ± SD. ANOVA was performed to compare groups after normality and variance equality were tested. Correlation was analyzed between blood oxygen saturation and the level of ET-1 and Ang II in the heart. A p value < 0.05 was considered statistically significant.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.In Group OSAHS, snoring and apnea progressed over time during sleep, and they showed interrupted sleep. There were no OSAHS-like symptoms in the control animals or in Group MAD. This finding suggested that OSAHS was successfully induced by palatal gel injection and that MADs were effective in alleviating the OSAHS-like symptoms in this study.p < 0.05) . It was also shown that there was a significant increase in AHI and a decrease in SaO2 in Group OSAHS (p < 0.05) by PSG, but there were no significant differences in AHI and SaO2 between Group MAD and the control group (p > 0.05)  Fig. . The retTo explore the pathophysiological damage to the heart caused by OSAHS, histological examinations were performed Fig. . In the The ultrastructure of the capillary among the myocardial fibers showed that the pinocytotic vesicles, mitochondria and rough surfaced endoplasmic reticulum were intact in the cytoplasm of endothelial cells in the control group Fig. . In GrouThe ultrastructure of myocardial fibers showed that the myofibers were well arranged and intercalated disks were clear in the control group. In Group OSAHS, the myofibril arrangement was disorderly, and the structure was blurred. Many myofibrils were dissolved, destroyed and had vanished. Some densified mitochondria and the fusion and disappearance of the cristae along with the membrane of mitochondria were present in the myocardial fibers in Group OSAHS. The myofibrillar arrangement was slightly disordered in Group MAD. A small amount of myocardial fibers collapsed and disappeared. Part of the myomere was unclear and unrecognizable. Local fusion and disappearance of the cristae along with the membrane of mitochondria were detected.a’ ratio was an estimate of left ventricular diastolic function, and the E/e’ ratio was an estimate of LV end-diastolic pressures. A decreased E/a’ ratio and increased E/e’ ratio were found in the OSAHS group, which suggested that the diastolic function of the left ventricle had degraded. However, there was no significant difference between Group MAD and the control group. LV systolic performance was assessed by the left ventricular ejection fraction (LVEF). Decreased diastolic function of the left ventricle was found in Group MAD and the control group. Left ventricular systolic performance was reduced in Group OSAHS, and there was no significant difference between Group MAD and the control group , a potent vasoconstrictor secreted from the endothelium, plays an important role in the pathogenesis of cardiovascular alterations30. Ang II is a multifunctional peptide that regulates vascular tone and blood pressure. Therefore, we speculated that OSAHS-induced myocardial injuries were associated with the overproduction of these systemic cytokines due to hypoxia. In this study, we first used ELISA and RT-PCR to examine the changes in the circulating levels of ET-1 and Ang II to explore their association with OSAHS-induced cardiovascular injury. In Group OSAHS, there was a significant increase in ET-1 and Ang II in plasma compared to the control group, while there was no significant difference between Group MAD and the control group. The level of ET-1 mRNA expression was higher in Group OSAHS than in Group MAD and the control group  could induce sympathetic nervous system overactivity, which resulted in the overproduction of angiotensin II (Ang II) and endothelin-1 (ET-1)oup Fig. . Second,oup Fig. . This su32, although it could not completely simulate all symptoms in OSAHS patients. Due to the limitations of clinical studies, OSAHS animal models provide useful tools to study the consequences of OSAHS and their underlying pathophysiological mechanisms. In addition, animal models could mimic some manifestations of OSAHS in humans under well-controlled experimental conditions. Therefore, in the present study, we used the OSAHS model developed in our previous study26. This model mimicked the characteristics of OSAHS to a large extent, including chronic intermittent hypoxia, sleep disruption, and increased-effort breathing.OSAHS is characterized by chronic intermittent hypoxia, sleep disruption and increased-effort breathing. The CIH model was most commonly used to investigate cardiovascular diseases caused by OSAHS2 by reduced nasal airflow, increased respiratory effort and sleep disruption in Group OSAHS. These findings were in accordance with the clinical features observed in OSAHS patients. However, all the OSAHS-like symptoms were alleviated after they were given MAD treatment, which suggested that the OSAHS model and MAD treatment were effective. In this study, we took advantage of the OSAHS animal model to examine the effects of OSAHS on the myocardium. It is well known that CPAP treatment of OSAHS patients has an impact on cardiovascular or cerebrovascular diseases such as hypertension33, coronary artery disease34, or cerebral infarct35. Although there was no definitive evidence to determine whether OSAHS directly caused cardiovascular disease, the data from this study implied that OSAHS could result in abnormal myocardium structure. This histological cardiac structure disorder and left ventricular hypotrophy in OSAHS rabbits were not detected in Group MAD, indicating that early treatment of MAD can prevent OSAHS-induced cardiovascular complications.Consistent with our previous studies, CBCT showed that the upper airway space was significantly decreased in Group OSAHS. This result suggested that gel injection induced upper airway stenosis successfully. The MADs protruded the mandible and enlarged the space of the retropalatal upper airway. PSG showed a significant increase in AHI and a decrease in SaOa’ and E/e’. It might be more convincing to evaluate overall cardiac dysfunction by a combined measure of left ventricular systolic and diastolic function in this study, rather than systolic or diastolic measures alone. Left ventricular systolic and diastolic dysfunction were found in OSAHS rabbits, and MAD treatment could rescue left ventricular systolic and diastolic function. The causes of left ventricular dysfunction included intermittent hypoxemia due to apnea or hypopnea, changes in left ventricular afterload and sleep fragmentation at night. Studies have shown that LV diastolic dysfunction and LV hypertrophy were detected in patients with OSAHS36. Furthermore, significantly elevated left ventricular end-systolic volume (LVESV) along with decreased LVEF were detected in a rat chronic intermittent hypopnea model37. However, there was no significant difference in LV function, although LV hypertrophy was found38. In addition, another study showed the beneficial effects of CPAP on the cardiac functions of patients with OSAHS39.In this study, echocardiographic images were recorded to determine the relative ventricular wall thickness. LV systolic performance was assessed by LVEF, and LV diastolic function was evaluated by E/The possible mechanisms of OSAHS-induced deleterious effects on the cardiovascular system may involve two major components. First, chronic intermittent hypoxia during repeated apnea or hypopnea resulted in sympathetic overdrive and endothelial cell dysfunction. Second, large negative intrathoracic pressures were generated during inspiratory efforts when apneas appeared, which increased transmural pressures across the myocardium and increased afterload, thus having a negative effect on myocardial structure and function. Additionally, frequent arousals from sleep led to increased sympathetic activity. LV systolic and diastolic function were significantly improved by MAD therapy. The beneficial effects of MADs may result from the following: the reopening of the constricted upper airway of OSAHS, consequently improving blood oxygen saturation, and the avoidance of the elevation of intrathoracic pressure, left ventricular afterload and sympathetic nerve activity. All of these factors could prevent the deleterious effects of OSAHS on cardiac structure and function.39. Sympathetic nervous system overactivity could result in the overproduction of Ang II and ET-1, which are important to vascular tone and blood pressure. CIH-induced cardiac dysfunction might be associated with decreased coronary tone. An intact coronary endothelium was shown to be important in regulating coronary blood flow during OSAHS8. Indeed, the level of ET-I and Ang II increased significantly in Group OSAHS compared with the control group and Group MAD, and there was a negative correlation of ET-1 and Ang II with blood oxygen saturation in the current study. This suggested that myocardium damage was probably due to the increased levels of ET-1 and Ang II in plasma, which were associated with intermittent hypoxia caused by OSAHS. First, intermittent hypoxia leads to damage to endothelial cells and functional disorders such that endothelial cells release too much ET-1. Second, hypoxia can increase the genetic transcription of ET-1 in endothelial cells40. Third, intermittent hypoxia may indirectly affect the synthesis41 and the secretion of ET-1 by affecting adrenaline and noradrenaline. Excess ET-1 binding specifically to ET receptor-A could stimulate local blood vessel contraction and extracellular calcium influx, which eventually resulted in the overload of intracellular calcium and damage to myocardial cells30. Ang II is mainly produced by endothelial cells and is the central part of the renin-angiotensin-aldosterone system (RAAS). It was reported that the levels of plasma renin activity (RA) and Ang II increased significantly in OSAHS patients and could be reduced through CPAP treatment, suggesting that OSAHS may increase RAAS activity29. On the other hand, myocardial damage could further exacerbate the hypoxia-increasing ET-I and Ang II levels in the plasma, and thus, a vicious circle is formed. Our findings suggested that early treatment of MADs could prevent serious cardiovascular complications caused by OSAHS.Sympathetic hyperactivity, oxidative stress and systemic inflammation have been regarded as potential mechanisms of hypertension and other cardiovascular diseases25. Second, it is hard to be test in animal study. In addition, CPAP can modify cardiac transmural pressure and therefore may directly influence cardiac functions42. Therefore, to test better tolerated MAD treatment is more practical. In this and our previous investigations26, we provided evidences that MAD normalized AHI and SaO2 status of OSAHS and did not change cardiac transmural pressure, it suggests that MAD remediated the adverse respiratory effects of OSAHS but not affecting the cardiac function in current model.Although CPAP is widely used to treat OSAHS and shows higher efficacy than MAD in treating OSAHS, it has some shortcomings. First, its efficacy is compromised by poor patient tolerance and complianceIn addition, we found that the cardiac dysfunctions and their alleviation discovered in this model is caused by respiratory airway relief, as we used Et1 and Ang II, which are correlated with average blood oxygen saturation, to evaluate their elevation in OSAHS and the reversing by MAD treatment. The results suggested that OSAHS-induced cardiac complications prevention is related to respiratory events prevention, and this is probably independent from the type of preventive upper airway obstruction mechanical treatment. Therefore, MAD can be recommended for severe OSAS treatment if supported with more clinical studies.